Mucoadhesive microorganism

ABSTRACT

The present disclosure provides genetically modified microorganisms (e.g., bacteria or yeast) with enhanced mucin-binding and/or cell-adhesion properties. For example, the present disclosure provides bacteria exhibiting increased in vitro binding to Caco-2 cells, and increased in vitro binding to mucins. Such microorganisms (e.g., bacteria) can be used, e.g., to deliver bioactive polypeptides to the gastrointestinal tract of a mammalian subject. Modifying the microorganism in the described manner allows for the modulation of gastrointestinal retention and transit times for the microorganism (e.g., bacterium). Exemplary microorganisms (e.g., lactic acid bacteria, such as  Lactococcus lactis ) contain an exogenous nucleic acid encoding a fusion protein containing a cell-adherence polypeptide, such as CmbA, and a mucin-binding polypeptide, such as a trefoil factor (TFF), e.g., human TFF3. The current disclosure further provides method for making and using the described microorganisms (e.g., bacteria).

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/IB2017/055470, filed Sep. 11, 2017, and claims benefit of the filingdate of U.S. Provisional Application No. 62/394,024, filed Sep. 13,2016.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 11, 2017, isnamed 205350-0031-00-WO_SL.txt and is 122,643 bytes in size.

BACKGROUND

Genetically modified microorganisms (e.g., bacteria) have been used todeliver therapeutic molecules to mucosal tissues. See, e.g., Steidler,L., et al., Nat. Biotechnol. 2003, 21(7): 785-789; Robert S. andSteidler L., Microb. Cell Fact. 2014, 13 Suppl. 1: S11; Braat et al.,Clin. Gastroenterol. Hepatol. 2006, 4(6):754-759; and Steidler et al.,Science 2000, 289(5483):1352-1355.

There is a need in the art for microbial (e.g., bacterial) strains withimproved pharmacokinetic and pharmacodynamics properties, and a need forefficacious, targeted, and controlled methods for the treatment ofvarious diseases treatable with such genetically modified bacteria. Thepresent disclosure addresses these needs.

BRIEF SUMMARY

The present disclosure provides microorganisms (e.g., bacteria or yeast)with enhanced cell-adhesion and/or mucin-binding properties. Forexample, the present disclosure provides bacteria exhibiting increasedin vitro binding to Caco-2 cells, and increased in vitro binding tomucins. Such microorganisms (e.g., bacteria) can be used, e.g., todeliver bioactive polypeptides to the gastrointestinal tract of amammalian subject, while the described genetic modifications allow forthe modulation of gastrointestinal retention and transit times of themicroorganism (e.g., bacterium). The described technology allows for themodulation of pharmacokinetic and pharmacodynamic properties of thebioactive polypeptides expressed by the microorganism (e.g., bacterium).For example, expression, secretion and anchoring of a fusion proteincontaining a trefoil factor (TFF) and a cell-adhesion polypeptide, suchas CmbA (see, e.g., Jensen et al., Microbiology 2014, 160(4):671-681)(e.g., TFF3-CmbA) in the cell wall of a lactic acid bacterium (LAB),such as Lactococcus lactis, enables adherence of the bacterium tointestinal epithelial cells, and further enables binding of thebacterium to mucins.

Compositions

In some aspects, the present disclosure provides microorganisms (e.g., abacteria or yeast) comprising an exogenous nucleic acid encoding afusion protein comprising a cell-adherence polypeptide. In someexamples, the current disclosure provides a microorganism (e.g.,bacterium or yeast) comprising an exogenous nucleic acid encoding afusion protein, wherein the exogenous nucleic acid encoding the fusionprotein contains a sequence encoding a cell-adherence polypeptide. Insome examples, the cell-adherence polypeptide is selected from the groupconsisting of cell and mucus-binding protein A (CmbA) (see, e.g., Jensenet al., Microbiology 2014, 160(4):671-681), mucus binding protein or mubdomain proteins (Mub) (see, e.g., Boekhorst et al., Microbiology 2006,152(1):273-280), mucus adhesion promoting protein (MapA) (see, e.g.,Miyoshi et al., Biosci. Biotechnol. Biochem. 2006, 70(7):1622-8),lactococcal mucin binding protein (MbpL) (see, e.g., Lukid et al., Appl.Environ. Microbiol. 2012, 78(22):7993-8000). A cell-wall anchoringpeptide, such as Staphylococcus aureus protein A anchor fragment (SpaX)may be added (see, e.g., Steidler et al., Appl. Environ. Microbiol.1998, 64(1):342-5). In some examples, the current disclosure provides amicroorganism (e.g., bacterium or yeast) comprising an exogenous nucleicacid encoding a fusion protein, wherein the fusion protein includes aCmbA polypeptide. In some examples, the CmbA polypeptide is CmbA fromLactobacillus reuteri. See, e.g., ATCC PTA6474, e.g., as disclosed inJensen et al., supra. In some examples according to any of the aboveembodiments, the cell-adherence polypeptide is a CmbA polypeptide havingan amino acid sequence that is at least 90%, at least 92%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% identical toSEQ ID NO: 1. In other examples according to any of the aboveembodiments, the cell-adherence polypeptide is a CmbA polypeptideencoded by an exogenous nucleic acid sequence that is at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to SEQ ID NO: 2.

In other examples according to any of the above embodiments, the fusionprotein comprises a mucin-binding polypeptide, such as a trefoil factor(TFF) polypeptide (e.g., TFF1, TFF2, or TFF3) or a MucBP polypeptide(see, e.g., Lukic et al, Appl. Environ. Microbiol. 2012,78(22):7993-8000). Thus, in some examples, the current disclosureprovides a microorganism (e.g., a bacterium or yeast) comprising anexogenous nucleic acid encoding a fusion protein, wherein the fusionprotein contains a cell-adherence polypeptide (e.g., a CmbA polypeptide)and a mucin-binding polypeptide (e.g., a TFF polypeptide). In someexamples, the TFF polypeptide is a human TFF polypeptide (e.g., hTFF1,hTFF2, or hTFF3). In some examples according to any of the aboveembodiments, the mucin-binding polypeptide is a human TFF3 polypeptidehaving an amino acid sequence that is at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 3. In other examples according to any of theabove embodiments, the mucin-binding polypeptide is a human TFF3polypeptide encoded by an exogenous nucleic acid sequence that is atleast 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 4. In some examples,the TFF polypeptide is a mammalian TFF polypeptide, such as a cow, pig,sheep, dog, cat, or horse TFF. In further examples, the TFF is anamphibian TFF. Exemplary TFF polypeptides are disclosed, e.g., in Conlonet al., Peptides 2015, 72:44-49, and cited references therein, each ofthe disclosures are incorporated herein by reference in their entirety.In other examples, the TFF polypeptide is a trefoil-like domain.Exemplary polypeptides according to this embodiment are disclosed inFujita et al., Mol. Reprod. Dev. 2006, 75(7):1217-1228, the disclosureof which is incorporated herein by reference in its entirety.

In some examples according to any of the above embodiments, the currentdisclosure provides a bacterium (e.g., a lactic acid bacterium, such asLactococcus lactis) comprising an exogenous nucleic acid encoding afusion protein comprising (1) a mucin-binding polypeptide selected froma TFF polypeptide (e.g., human TFF1, human TFF2, or human TFF3) and aMucBP polypeptide; and (2) a cell-adherence polypeptide selected from aCmbA polypeptide, a Mub polypeptide, a MapA polypeptide, an MbpLpolypeptide, and a SpaX polypeptide. In some examples according to thisembodiment, the fusion protein contains a CmbA polypeptide (e.g.,Lactobacillus reuteri CmbA) and a TFF polypeptide (e.g., human TFF1,human TFF2, or human TFF3).

In some examples according to any of the above embodiments, theexogenous nucleic acid encoding the fusion protein is integrated intothe chromosome of the microorganism, e.g., the chromosome of abacterium. In some examples, the exogenous nucleic acid encoding thefusion protein is constitutively expressed in the microorganism (e.g.,bacterium). In other examples, the exogenous nucleic acid encoding thefusion protein is located on a plasmid.

In some examples according to any of the above embodiments, the fusionprotein is expressed by the microorganism (e.g., bacterium). In otherexamples the fusion protein is anchored in the cell wall of themicroorganism (e.g., bacterium). For example, the fusion protein isdisplayed on the surface (i.e., outer membrane) of the microorganism(e.g., bacterium).

In some examples according to any of the above embodiments, theexogenous nucleic acid encoding a fusion protein further includes asecretion leader sequence encoding a secretion signal peptide. In someexamples, the secretion leader sequence contains a nucleotide sequenceencoding a secretion leader of unidentified secreted 45-kDa protein(Usp45). Such secretion leader sequence or peptide is referred to hereinas SSusp45. In some examples, SSusp45 has an amino acid sequence that isat least 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 5. In other examples,SSusp45 is encoded by a nucleic acid sequence that is at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to SEQ ID NO: 6 or SEQ ID NO: 7. Any secretionleader sequence derived from a gram-positive bacterium, e.g., anysecretion leader sequence derived from Lactococcus lactis is useful inthe context of the above embodiments. In further examples according toany of the above embodiments, the secretion signal peptide (e.g.,SSusp45) is bound to the mucin-binding polypeptide, such as such as aTFF polypeptide. In some examples according to this embodiment, SSusp45is bound to a human TFF polypeptide. For examples, the fusion proteinmay include an amino acid sequence that is at least 90%, at least 92%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 8. In other examples, the fusion protein may beencoded by an exogenous nucleic acid sequence containing a sequence thatis at least 90%, at least 92%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identical to SEQ ID NO: 9.

In other examples, the microorganism (e.g., bacterium) comprises anexogenous nucleic acid encoding a fusion protein containing a TFFpolypeptide and a CmbA polypeptide, wherein a secretion signal peptideis bound to the TFF polypeptide (e.g., SSusp45). For example, the fusionprotein may include (or consist of) an amino acid sequence that is atleast 90%, at least 92%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO: 10. In otherexamples, the fusion protein may be encoded by an exogenous nucleic acidsequence containing (or consisting of) a sequence that is at least 90%,at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identical to SEQ ID NO: 11.

In some examples, the secretion signal peptide includes a linkerpeptide. In some examples, the secretion signal peptide is cleaved fromthe fusion protein, e.g., when the fusion protein is anchored in a cellwall of the microorganism (e.g., bacterium).

In some examples, the exogenous nucleic acid encoding the fusion proteinis transcriptionally regulated by (e.g., placed under the control of) apromoter that is endogenous to the microorganism (e.g., bacterium). Inother examples, expression of the fusion protein is controlled by anexogenous promoter. In some examples, the promoter is selected from athyA promoter (PthyA), an hlla promoter (PhllA), and a gapB promoter. Insome examples, the nucleic acid encoding a fusion protein istranscriptionally regulated by a PthyA promoter. In other examples, theexogenous nucleic acid encoding the fusion protein is transcriptionallyregulated by a PhllA promoter. Other promoters include those precedinggenes holA, soda, enoA, tufa, fbaA, acpA, ps431, malG, ptsH, dpsA, pgk,ahpC, pdhD, pts_II, pfk, trePP, ptnD, pgiA, usp45. Other suitablepromoters are described, e.g., in U.S. Patent Application Publication2014/0105863, the disclosure of which is incorporated herein byreference in its entirety.

The present disclosure further provides a microorganism (e.g.,bacterium) comprising a fusion protein (e.g., anchored in a cell-wall ofthe microorganism, e.g., bacterium), wherein the fusion proteincomprises a TFF polypeptide and a CmbA polypeptide. In some examples,the microorganism (e.g., bacterium) includes an exogenous nucleic acidcomprising a secretion leader sequence, a sequence encoding the TFFpolypeptide, and a sequence encoding the CmbA polypeptide. In someexamples, the secretion leader sequence encodes a secretion signalpeptide, which is cleaved from the fusion protein, e.g., when the fusionprotein passes the cytoplasmic membrane (e.g., is anchored in the cellwall) of the microorganism (e.g., bacterium).

In some examples according to any of the above embodiments, themicroorganism is a bacterium. In other examples according to any of theabove embodiments, such bacterium is a Gram-positive bacterium, e.g., anon-pathogenic Gram-positive bacterium. In other examples according toany of the above embodiments, the bacterium is a lactic acid bacterium(LAB). Exemplary lactic acid bacteria are disclosed herein, each ofwhich can be used in the context of these embodiments. In someembodiments, the LAB is selected from the group consisting of aLactococcus species (sp.) bacterium, a Lactobacillus sp. bacterium, aBifidobacterium sp. bacterium, a Streptococcus sp. bacterium, and anEnterococcus sp. bacterium. In some examples, the LAB is Lactococcuslactis. In other examples, the LAB is selected from Lactococcus lactissubsp. cremoris, Lactococcus lactis subsp. hordniae, and Lactococcuslactis subsp. lactis. In some examples, the Lactococcus lactis isLactococcus lactis subsp. cremoris, such as Lactococcus lactis strainMG1363.

In some examples according to any of the above embodiments, themicroorganism (e.g., bacterium) comprises an exogenous nucleic acidencoding a fusion protein comprising a TFF polypeptide and acell-adherence polypeptide (e.g., CmbA). In some examples according tothis embodiment, the TFF polypeptide is selected from TFF1, TFF2, andTFF3. In other examples according to this embodiment, the TFFpolypeptide is selected from human TFF, mouse TFF, pig TFF, dog TFF, catTFF, cow TFF, and sheep TFF. In some examples, the TFF polypeptide ishuman TFF. In other examples, the TFF polypeptide is selected from humanTFF1, human TFF2, and human TFF3. In yet other example, the TFFpolypeptide is human TFF3. In some example, the TFF polypeptide has anamino acid sequence at least 90%, at least 92%, at least 95%, at least96%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In otherexamples, the TFF polypeptide is a TFF variant polypeptide, e.g., a TFFvariant polypeptide having enhanced mucin-binding capability whencompared to a corresponding wild-type TFF polypeptide. In otherexamples, the TFF polypeptide is an amphibian or fish TFF polypeptide.

In some examples according to any of the above embodiments, themicroorganism (e.g., bacterium) further contains an exogenous nucleicacid encoding at least one therapeutic polypeptide. In some examples,the therapeutic polypeptide is a cytokine, such as an interleukin (IL).The choice of cytokine is made on the basis of what host responses aresought to be activated or suppressed. In some examples, the cytokine isIL-2, IL-10, or IL-22. In other examples, the therapeutic polypeptide isan antigen. In other examples the therapeutic polypeptide is an antigenand an interleukin, such as IL-2, IL-10, or IL-22. In some examplesaccording to any of these embodiments, the antigen is an autoantigen,e.g., a T1D-specific antigen. Exemplary T1D-specific antigens includeproinsulin (PINS), glutamic acid decarboxylase (GAD65),insulinoma-associated protein 2 (IA-2), islet-specificglucose-6-phosphatase catalytic subunit-related protein (IGRP), zinctransporter 8 (ZnT8), chromogranin A, (prepro) islet amyloid polypeptide(ppIAPP), peripherin, citrullinated glucose-regulated protein (GRP), anda combinations thereof. Exemplary amino acid sequences and nucleic acidsequences for the above T1D-specific antigens are disclosed, e.g., ininternational patent application publication WO2017/122180, thedisclosure of which is incorporated herein by reference in its entirety.In other examples, the antigen is an allergen, such as a tree pollenallergen, a weed pollen allergen, a grass pollen allergen, a foodallergen, a dust-mite allergen, a mold allergen, an animal danderallergen, or a combination thereof. In some examples, the allergen is aweed pollen allergen, e.g., a ragweed pollen allergen. In otherexamples, the allergen is a tree pollen allergen, such as a birch pollenallergen or a Japanese cedar pollen allergen. In yet other examples, theallergen is a food allergen, such as a peanut allergen, a milk allergen,an egg allergen, a gluten allergen (gliadin epitope), or a combinationthereof.

In further examples, the therapeutic polypeptide is an antibody or afragment thereof. For example, the antibody is a single-domain antibody(e.g. camelid or shark antibody) or a nanobody. Exemplary antibodiesinclude cytokine neutralizing antibodies such as antibodies to IL-4,antibodies to IL-5, antibodies to IL-7, antibodies to IL-13, antibodiesto IL-15, as well as anti TNFα antibodies to immunoglobulin E (IgE), andany fragments thereof. In some examples, the therapeutic polypeptide isa fusion protein. For example, the therapeutic polypeptide comprises asoluble receptor, such as a TNF receptor (e.g., soluble TNF receptor 2)and an antibody or an antibody fragment, such as the Fc region of anantibody. In some examples according to these embodiments, thetherapeutic polypeptide contains an Fc region of a human immunoglobulin(e.g., human IgG1 Fc). In some examples, the therapeutic polypeptidecomprises soluble TNF receptor 2 fused to human IgG1 Fc). In someexamples, the therapeutic polypeptide is etanercept.

In yet other examples, the therapeutic polypeptide is an enzyme or afragment (e.g., functional fragment) thereof, e.g., a phenylalanineammonia lyase (PAL), an amino acid decarboxylase, or a combinationthereof. In one example, the therapeutic polypeptide is PAL, or afunctional fragment thereof.

In further examples, the therapeutic polypeptide is a glucagon-likepeptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), glucagon, exendin-4,or any combination thereof. In other examples, the therapeuticpolypeptide is a growth factor, such as an epidermal growth factor(EGF), e.g., human EGF or porcine EGF. In yet other examples, thetherapeutic polypeptide is a TFF, such as TFF1, TFF2, TFF3, or acombination thereof.

The therapeutic polypeptide may be a combination of any of the aboverecited therapeutic polypeptides.

In some examples according to any of the above embodiments, theexogenous nucleic acid encoding the at least one therapeutic polypeptideis transcriptionally regulated by a promoter selected from a gapBpromoter (PgapB), a thyA promoter (PthyA), and an h/la promoter (PhllA).In some examples, the exogenous nucleic acid encoding the at least onetherapeutic polypeptide is transcriptionally regulated by (e.g., underthe control of) a gapB promoter. Other promoters include those precedinggenes holA, soda, enoA, tufa, fbaA, acpA, ps431, malG, ptsH, dpsA, pgk,ahpC, pdhD, pts_II, pfk, trePP, ptnD, pgiA, usp45. Other suitablepromoters are described, e.g., in U.S. Patent Application Publication2014/0105863, the disclosure of which is incorporated herein byreference in its entirety.

In some examples according to any of the above embodiments, themicroorganism (e.g., LAB) further comprises a combination of mutationsand insertions to promote trehalose accumulation, which enhances LABsurvivability against bile salts and drying. For example, these may be

(i) chromosomally-integrated trehalose transporter(s), such asPhllA>>transporter 1>>intergenic region>>transporter 2, such asllmg_0453 and/or llmg_0454, for uptake of trehalose;

(ii) chromosomally-integrated Trehalose-6-phosphate phosphatase gene(otsB; Gene ID: 1036914) positioned downstream of usp45 (Gene ID:4797218) to facilitate conversion of trehalose-6-phosphate to trehalose;

(iii) inactivated (e.g., through gene deletion) Trehalose-6-phosphatephosphorylase gene (trePP; Gene ID: 4797140); and

(iv) inactivated cellobiose-specific PTS system IIC component (Gene ID:4796893), ptcC, (e.g., tga at codon position 30 of 446; tga30).

For example, an exogenous nucleic acid encoding a trehalose-6-phosphatephosphatase, e.g., otsB, such as Escherichia coli otsB. In some examplesaccording to these embodiments, the exogenous nucleic acid encoding thetrehalose-6-phosphate phosphatase is chromosomally integrated. In someexamples, the exogenous nucleic acid encoding the trehalose-6-phosphatephosphatase is chromosomally integrated 3′ of unidentified secreted45-kDa protein gene (usp45). In some examples according to thisembodiment, the LAB comprises a second polycistronic expression cassettecomprising a usp45 promoter, the usp45 gene (e.g., 3′ of the promoter),and the exogenous nucleic acid encoding a trehalose-6-phosphatephosphatase (e.g., 3′ of the usp45 gene). In some examples, the secondpolycistronic expression cassette further comprises an intergenic regionbetween the usp45 gene and the exogenous nucleic acid encoding atrehalose-6-phosphate phosphatase. In some examples, the secondpolycistronic expression cassette is illustrated by:Pusp45>>usp45>>intergenic region>>otsB. In some examples according tothese embodiments, the intergenic region is rpmD as described hereinabove (e.g., having SEQ ID NO: 8 or SEQ ID NO: 9). The secondpolycistronic expression cassette may then be illustrated by:Pusp45>>usp45>>rpmD>>otsB.

In some examples according to any of the above embodiments, atrehalose-6-phosphate phosphorylase gene (trePP) is disrupted orinactivated in the microorganism (e.g., LAB). For example, the trePP hasbeen inactivated by removing the trePP gene or a fragment thereof, orthe trePP has been disrupted by inserting a stop codon. Thus, in someexamples according to these embodiments, the microorganism (e.g., LAB)lacks trePP activity.

In other examples according to any of the above embodiments, acellobiose-specific PTS system IIC component gene (ptcC) has beendisrupted or inactivated in the microorganism (e.g., LAB). For example,the ptcC has been disrupted by inserting a stop codon, or ptcC has beeninactivated by removing the ptcC or a fragment thereof. Thus, in someexamples according to these embodiments, the microorganism (e.g., LAB)lacks ptcC activity.

In other examples according to any of the above embodiments, the LABfurther comprises one or more genes encoding one or more trehalosetransporter(s). In some examples, the one or more genes encoding the oneor more trehalose transporter(s) are endogenous to the LAB. In someexamples, the LAB overexpresses the one or more genes encoding the oneor more trehalose transporter(s). In some examples according to theseembodiments, the one or more genes encoding the one or more trehalosetransporter(s) is positioned 3′ of an exogenous promoter, e.g., an hllApromoter (PhllA). For example, the one or more genes encoding the one ormore trehalose transporter(s) are transcriptionally regulated by thePhllA. In some examples according to these embodiments, the one or moregenes encoding the one or more trehalose transporter(s) is selected fromllmg_0453, llmg_0454, and any combination thereof. In some examples, 11mg 0453 and llmg_0454 are transcriptionally regulated by a PhllA.

In some examples, according to any of the above embodiments, the one ormore genes encoding one or more trehalose transporter(s) comprises twogenes encoding two or more trehalose transporters, wherein an intergenicregion is located between the two genes. In some examples, theintergenic region is rpmD, e.g., having SEQ ID NO: 8 or SEQ ID NO: 9. Insome examples, the microorganism (e.g., LAB) comprises a polycistronicexpression cassette comprising two nucleic acid sequences (e.g., genes)encoding two different trehalose transporters (transporter 1 andtransporter 2 sequences) and an intergenic region between the twonucleic acids encoding the two different trehalose transporters. Suchexpression cassette may be illustrated by: PhllA>>transporter1>>intergenic region>>transporter 2. In some examples according to theseembodiments, the intergenic region is rpmD as described herein above(e.g., having SEQ ID NO: 8 or SEQ ID NO: 9). The polycistronicexpression cassette may then be illustrated by:PhllA>>transporter1>>rpmD>>transporter2.

Thus, in some embodiments, the LAB comprises, in a single strain,several useful features. In one embodiment, the LAB is Lactococcuslactis, comprising:

-   (A) a chromosomally integrated promoter>>secretion signal>>mucin and    cell adherence fusion protein;-   (B) one or more of a chromosomally-integrated promoter>>secretion    signal>>therapeutic protein;-   (C) a combination of mutations and insertions to promote trehalose    accumulation, which enhances LAB survivability against bile salts    and drying. The mutations are selected from    -   (i) chromosomally-integrated trehalose transporter(s), such as        PhllA>>transporter 1>>intergenic region>>transporter 2, such as        llmg_0453 and/or llmg_0454, for uptake of trehalose;    -   (ii) chromosomally-integrated Trehalose-6-phosphate phosphatase        gene (otsB; Gene ID: 1036914) positioned downstream of usp45        (Gene ID: 4797218) to facilitate conversion of        trehalose-6-phosphate to trehalose;    -   (iii) inactivated (e.g. through gene deletion)        Trehalose-6-phosphate phosphorylase gene (trePP; Gene ID:        4797140); and    -   (iv) inactivated cellobiose-specific PTS system IIC component        (Gene ID: 4796893), ptcC, (e.g. tga at codon position 30 of 446;        tga30).        The LAB may also contain an auxotrophic mutation for biological        containment, such as thyA. One or more of these functions (i.e.        mucin and cell adherence, therapeutic protein, trehalose        accumulation) may be in a polycistronic operon, wherein each        gene may be separated by an intergenic region.

In one embodiment, the LAB is Lactococcus lactis, with

-   (A) thyA mutation, for biological containment-   (B) a chromosomally integrated PthyA>>SSusp45-hTFF3-cmbA to export    and link to the cell wall a mucus and cell binding fusion protein.-   (C) a chromosomally-integrated PgapB>>gapB>>intergenic region (such    as rpmD)>>SSusp45>>pal, to secrete mature PAL from LAB;-   (D) chromosomally-integrated trehalose transporter(s), such as    PhllA>>transporter 1>>intergenic region>>transporter 2, such as    llmg_0453 and/or llmg_0454, for uptake of trehalose;-   (E) inactivated (e.g. through gene deletion) Trehalose-6-phosphate    phosphorylase gene (trePP; Gene ID: 4797140);-   (F) chromosomally integrated Trehalose-6-phosphate phosphatase gene    (otsB; Gene ID: 1036914) (positioned downstream of usp45 (Gene    ID: 4797218) to facilitate conversion of trehalose-6-phosphate to    trehalose; and-   (G) inactivated cellobiose-specific PTS system IIC component (Gene    ID: 4796893), ptcC, (e.g. tga at codon position 30 of 446; tga30).

In some examples according to any of the above embodiments, themicroorganism (e.g., bacterium) has an increased gastro-intestinal (GI)transit time when compared to a corresponding microorganism (e.g.,bacterium) not comprising the described genetic modification, i.e., notcomprising said exogenous nucleic acid encoding the fusion protein ornot comprising said fusion protein. In some examples, the GI transittime is increased by at least about 10%, at least about 30%, at leastabout 50%, at least about 80%, or at least about 100% (to about 2×). Inother examples, the GI transit time is increased from at least about 10%to about 500%, from at least about 20% to about 400%, from at leastabout 20% to about 300%, from at least about 20% to about 300%, or fromat least about 30% to about 300%.

In some examples according to any of the above embodiments, themicroorganism (e.g., bacterium) exhibits increased in vitromucin-binding capability when compared to a corresponding microorganism(e.g., bacterium) not genetically modified as described herein, i.e.,not comprising an exogenous nucleic acid encoding a fusion protein ornot comprising a fusion protein. In some examples, the in vitromucin-binding capability is increased by at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90% or at leastabout 100% (to about 2×). In other examples, the mucin-bindingcapability is increased from at least about 10% to about 500%, from atleast about 10% to about 400%, from at least about 10% to about 300%,from at least about 10% to about 200%, from at least about 20% to about200%, from at least about 20% to about 300%, or from at least about 20%to about 500%.

Mucin-binding capabilities can be measured in accordance with anyart-recognized method, e.g., those described herein. In some examples,in vitro mucin-binding capability is measured by contacting and bindingthe microorganism (e.g., a bacterium) to immobilized mucins (e.g.,mucins from porcine stomach), and measuring the number of microbialcells (e.g., bacterial cells) bound to the mucin, e.g., by detectinglight absorbance at an appropriate wavelength, e.g., at 405 nm (OD₄₀₅);or by staining the bound microbial cells (e.g., bound bacterial cells)with a dye (e.g., crystal violet) and subsequently detecting lightabsorbance at a wavelength appropriate for the employed dye. Forexample, if crystal violet is used to stain bacterial cells bound to themucin, light absorbance may be measured at 595 nm (OD₅₉₅).

In some examples according to any of the above embodiments, themicroorganism (e.g., bacterium) exhibits increased in vitro Caco-2cell-binding capability when compared to a corresponding microorganism(e.g., bacterium) without the described genetic modification, i.e., notcomprising an exogenous nucleic acid encoding the fusion protein, or notcomprising the fusion protein. In some examples, the in vitro Caco-2binding capability is increased by at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 80%, at least about 100% (to about 2×),at least about 200%, at least about 300%, or at least about 400% (about5×). In other examples, the in vitro Caco-2 binding capability isincreased from at least about 10% to about 200%, from at least about 10%to about 300%, from at least about 10% to about 400%, or from at leastabout 10% to about 500%. Caco-2 cell binding capability can be measuredin accordance with any art-recognized method, such as those disclosedherein. For example, Caco-2 binding capability is measured by: (i)contacting the microorganism (e.g., bacterium), e.g., a culture of themicroorganism (e.g., bacterium) with Caco-2 cells; (ii) washing theCaco-2 cells to remove unbound microbial (e.g., bacterial cells); (iii)detaching the microbial cells (e.g., bacterial cells) that are bound tothe Caco-2 cells; and (iv) determining the number of detached bacterialcells (i.e., titering the detached cells), e.g., as described herein.

In some examples according to any of the above embodiments, themicroorganism (e.g., bacterium) exhibits increased adherence tointestinal mucosa when compared to a corresponding microorganism (e.g.,bacterium) without the described genetic modification, i.e., notcomprising an exogenous nucleic acid encoding the fusion protein or notcomprising the fusion protein. In some examples, adherence of themicroorganism (e.g., bacterium) to intestinal mucosa is increased fromat least about 10% to about 100% (to about 2×), from at least about 10%to about 200%, from at least about 10% to about 400%, or from at leastabout 10% to about 500%.

The present disclosure further provides a composition comprising amicroorganism (e.g., a bacterium) of the present disclosure, e.g., amicroorganism (e.g., bacterium) as described in any of the aboveembodiments. For example, the present disclosure provides a compositioncomprising a microorganism (e.g., a bacterium) comprising a fusionprotein (e.g., anchored in a cell-wall of the microorganism, e.g.,bacterium), wherein the fusion protein comprises a TFF polypeptide and aCmbA polypeptide. In some examples, the microorganism (e.g., bacterium)includes an exogenous nucleic acid comprising a secretion leadersequence (e.g., SSusp45), a sequence encoding the TFF polypeptide, and asequence encoding the CmbA polypeptide. In some examples, the secretionleader sequence encodes a secretion signal peptide, which is cleavedfrom the fusion protein, e.g., when the fusion protein is passing thecytoplasmic membrane of the bacterium.

The present disclosure further provides a pharmaceutical compositioncomprising a microorganism (e.g., a bacterium) of the present disclosureand a pharmaceutically acceptable carrier. For example, thepharmaceutical composition contains a microorganism (e.g., a bacterium)as described in any of the above embodiments.

The present disclosure further provides a microorganism (e.g.,bacterium) of the present disclosure, or a composition (e.g., apharmaceutical composition) of the present disclosure for use in thetreatment of a disease. The present disclosure further provides amicroorganism (e.g., a bacterium) or a composition (e.g., apharmaceutical composition) for use in the preparation of a medicamentfor the prevention or treatment of a disease. In some examples accordingto any of these embodiments, the disease is selected from an autoimmunedisease, an allergy, a nutritional or metabolic disease, agastro-intestinal disease, and a genetic disorder. Further diseases thatcan be treated using the microorganisms (e.g., bacteria) andcompositions of the present disclosure are described herein.

The present disclosure further provides an isolated nucleic acidencoding a fusion protein, said nucleic acid comprising: (i) a sequenceencoding a mucin-binding polypeptide, such as a TFF polypeptide (e.g.,TFF1, TFF2, or TFF3) or a MucBP polypeptide; and (ii) a sequenceencoding a cell-adherence polypeptide, such as a CmbA polypeptide, a Mubpolypeptide, a MapA polypeptide, or a MbpL polypeptide. In some examplesaccording to these embodiments, the cell-adherence polypeptide is a CmbApolypeptide. Accordingly, the present disclosure provides an isolatednucleic acid encoding a fusion protein, wherein the nucleic acidcontains (i) a sequence encoding a TFF polypeptide, such as TFF1 (e.g.,human TFF1), TFF2 (e.g., human TFF2), or TFF3 (e.g., human TFF3), and(ii) a sequence encoding a CmbA polypeptide.

The present disclosure further provides a plasmid comprising theisolated nucleic acid of the present disclosure, e.g., an isolatednucleic acid in accordance with any of the above embodiments.

The present disclosure further provides a microbial (e.g., bacterial)host cell comprising an isolated nucleic acid of the present disclosureor a plasmid of the present disclosure.

The present disclosure further provides a kit comprising (1) amicroorganism (e.g., a bacterium), a composition, a pharmaceuticalcomposition, or a unit dosage form of the present disclosure; and (2)instructions for administering the microorganism (e.g., bacterium), thecomposition, the pharmaceutical composition, or the unit dosage form toa mammal, such as an animal or human subject or patient.

Methods

The present disclosure further provides methods for the treatment of adisease in a subject in need thereof. Exemplary methods include:administering to the subject a therapeutically effective amount of amicroorganism (e.g., a bacterium), a composition, or a pharmaceuticalcomposition of the present disclosure. In some examples according tothese embodiments, the microorganism (e.g., bacterium) comprises anexogenous nucleic acid encoding a therapeutic polypeptide as describedherein. Exemplary diseases that can be treated using such microorganisms(e.g., bacteria) and compositions of the present disclosure aredescribed herein. In some examples, the disease is an autoimmunedisease, an allergy, a nutritional or a metabolic disease, agastro-intestinal disease, a genetic disorder, or any combinationsthereof. In some examples according to any of the above embodiments, thedisease is an autoimmune disease, such as type-1 diabetes (T1D). Inother examples, the disease is a metabolic disease, such asphenylketonuria (PKU). In other examples, the disease is agastro-intestinal disease, such as celiac disease, or inflammatory boweldisease (IBD), e.g., Crohn's disease or ulcerative colitis. In furtherexamples, the disease is growth retardation.

In some embodiments, the disease is phenylketonuria (PKU). In someexamples according to this embodiment, the microorganism (e.g.,bacterium) comprises an exogenous nucleic acid encoding a polypeptide,e.g., an enzyme that is capable of degrading phenylalanine (Phe), e.g.,in the GI tract, e.g., prior to absorption of the Phe by the subject towhich the microorganism is being administered. In some examplesaccording to these embodiments, the microorganism (e.g., bacterium)comprises an exogenous nucleic acid encoding a phenylalanine ammonialyase (PAL), an enzyme that converts Phe to cinnamic acid. Thus, thepresent disclosure provides methods for the treatment of PKU in asubject in need thereof. Exemplary methods include: administering to thesubject a therapeutically effective amount of a microorganism (e.g., abacterium), a composition, or a pharmaceutical composition of thepresent disclosure, wherein the microorganism (e.g., bacterium)comprises an exogenous nucleic acid encoding PAL. Inhibition of Pheabsorption and treatment of PKU may be analyzed using a mouse PKU model,e.g., utilizing (enu2/2) mice (see, e.g., Sarkissian, C. N. et al.,Proc. Natl. Acad Sci. USA 1999, 96: 2339-2344), or using a rat model(see, e.g., Chang et al., Artif. Cells Blood Substit. Immobil.Biotechnol. 1995, 23(1):1-21).

The present disclosure further provides methods for preparing agenetically modified microorganism (e.g., bacterium). Exemplary methodsinclude: contacting an exogenous nucleic acid encoding a fusion proteinwith a microorganism (e.g., bacterium), wherein the exogenous nucleicacid encoding the fusion protein comprises a sequence encoding acell-adherence polypeptide, such as a CmbA polypeptide, a Mubpolypeptide, a MapA polypeptide, or a MbpL polypeptide. In someexamples, the cell-adherence polypeptide is CmbA, e.g., Lactobacillusreuteri CmbA. In some examples, carrying out the above method (i.e.,contacting the microorganism with the exogenous nucleic acid) results ina microorganism (e.g., bacterium) containing the exogenous nucleic acidencoding the fusion protein. In other examples, contacting themicroorganism (e.g., bacterium) with the exogenous nucleic acid resultsin a microorganism (e.g., bacterium) containing the exogenous nucleicacid encoding the fusion protein, and thereby expressing the fusionprotein encoded by the exogenous nucleic acid. In some examples, themethod further includes culturing the microorganism (e.g., bacterium)and expressing the fusion protein in the microorganism (e.g.,bacterium). In some examples, the contacting occurs under conditionssufficient for said bacterium to internalize the exogenous nucleic acid.In some examples according to any of these embodiments, the exogenousnucleic acid is located on a plasmid. In some examples according tothese embodiments, the exogenous nucleic acid is integrated into thechromosome of the bacterium.

In some examples according to any of the above embodiments, theexogenous nucleic acid encoding the fusion protein further comprises asequence encoding a mucin-binding polypeptide, such as a trefoil factor(TFF) polypeptide or a MucBP polypeptide. In some examples, theexogenous nucleic acid encoding the fusion protein comprises a sequenceencoding CmbA and a sequence encoding a TFF polypeptide. Accordingly,the present disclosure provides a method for preparing a geneticallymodified microorganism (e.g., bacterium) comprising: contacting anexogenous nucleic acid encoding a fusion protein with a microorganism(e.g., bacterium), wherein the exogenous nucleic acid encoding thefusion protein comprises (i) a sequence encoding a TFF polypeptide(e.g., TFF1, TFF2, or TFF3) and (ii) a sequence encoding CmbA. In someexamples according to any of these embodiments, the exogenous nucleicacid encoding the fusion protein is chromosomally integrated (e.g.,integrated into the chromosome of a bacterium), e.g., by usinghomologous recombination. In accordance with this embodiment, the methodcan further include forming a plasmid (i.e., an integration plasmid)comprising the exogenous nucleic acid encoding the fusion protein.

In one example according to any of the above embodiments, the methodfurther includes contacting the microorganism (e.g., bacterium) with anexogenous nucleic acid encoding a therapeutic polypeptide, e.g., priorto or subsequent to contacting the microorganism (e.g., bacterium) withan exogenous nucleic acid encoding the fusion protein.

In some examples according to any of the above embodiments, thegenetically modified microorganism (e.g., bacterium) prepared by theabove methods exhibits increased muco- and/or cell-adhesive propertiesas described herein when compared to a corresponding microorganism(e.g., bacterium) not modified according to the instant method, i.e.,not comprising an exogenous nucleic acid encoding the fusion protein.

In related embodiments, the adhesion to mucus and/or cells is specificto types of mucus and/or cells. As a result of preferential binding tospecific receptors found in specific cells or mucus, the bacterium maybe localized to specific sites. In such a way, it is possible to ensurelocalization of the bacterium to the site where delivery of specificmolecules is most effective. In some embodiments that location may bethe mucosae (intestine, oral cavity, eye, ear, urogenital). In someembodiments that location may be the small bowel, in some embodimentsthat location may be the upper small bowel.

In some examples, the method further includes combining a culture of thegenetically modified bacterium with at least one cryopreserving agent toform a bacterial mixture. The method may further include drying (e.g.,freeze-drying or spray drying) the bacterial mixture to form a dried(e.g., freeze-dried) composition. The method can further includecombining the genetically modified bacterium, or the dried composition(e.g., freeze-dried composition) with a pharmaceutically acceptablecarrier to form a pharmaceutical composition. The method may furtherinclude formulating the genetically modified bacterium, the dried (e.g.,freeze-dried) composition, or the pharmaceutical composition into apharmaceutical unit dosage form, such as a tablet, capsule, or granule.

The current disclosure further provides a unit dosage form comprising atleast one microorganism (e.g., a bacterium), a composition, or apharmaceutical composition of the present disclosure. In some examples,such unit dosage form is an oral dosage form. In other examplesaccording to these embodiments, the unit dosage form is a capsule (e.g.,a capsule containing a powder or containing micro-pellets ormicro-granules), a tablet, a granule, a sachet, or a packaged liquid,e.g., suspension. In other embodiments, the unit dosage form is ametered aerosol dose, or a suppository.

In some embodiments, the microorganism (e.g., the non-pathogenicGram-positive bacterium) contained in the dosage form is in a dry-powderform or a compacted version thereof.

The current disclosure further provides a unit dosage form comprisingfrom about 1×10⁴ to about 1×10¹² colony-forming units (cfu) of amicroorganism of the present disclosure, e.g., a non-pathogenicmicroorganism (e.g., a non-pathogenic Gram-positive bacterium). In someembodiments, the unit dosage form comprises from about 1×10⁶ to about1×10¹² colony-forming units (cfu) of the microorganism (e.g., thenon-pathogenic Gram-positive bacterium). In other embodiments, the unitdosage form comprises from about 1×10⁹ to about 1×10¹² colony-formingunits (cfu) of the microorganism (e.g., the non-pathogenic Gram-positivebacterium).

In some embodiments in any of the above methods, the microorganism(e.g., bacterium) is administered to a subject orally. For example, themicroorganism (e.g., bacterium) is administered to the subject in theform of a pharmaceutical composition for oral administration (e.g., acapsule, tablet, granule, suspension or liquid) comprising themicroorganism (e.g., bacterium) and a pharmaceutically acceptablecarrier. In other examples, the microorganism (e.g., bacterium) isadministered to the subject in the form of a food product, or is addedto a food (e.g., a drink). In other examples, the microorganism (e.g.,bacterium) is administered to the subject in the form of a dietarysupplement. In yet other examples, the microorganism (e.g., bacterium)is administered to the subject in the form of a suppository product. Insome examples, the compositions of the present disclosure are adaptedfor mucosal delivery of the polypeptides, which are expressed by themicroorganism (e.g., bacterium). For example, compositions may beformulated for efficient release of a therapeutic polypeptide in theintestinal tract of the subject.

The current disclosure further provides a genetically modifiedbacterium, a composition, a pharmaceutical composition, or a unit dosageform prepared by a method in accordance with any of the aboveembodiments.

The present disclosure further provides methods for enhancing growth ina mammal. Exemplary methods include administering to the mammal aneffective amount of a microorganism (e.g., bacterium), a composition, apharmaceutical composition, or a unit dosage form of the presentdisclosure. In some examples, the mammal is a human, a farm animal(e.g., a pig, a cow, a goat, or a sheep), a dog, a cat, or otherdomestic animal. In some examples, the microorganism (e.g., bacterium),the composition, pharmaceutical composition, or the unit dosage form isformulated for administration to the mammal, e.g., is formulated fororal administration. In some examples, the microorganism (e.g.,bacterium) employed in this method, contains an exogenous nucleic acidencoding a growth factor or growth hormone. In some examples accordingto this embodiment, the growth factor or growth hormone isconstitutively expressed in the microorganism (e.g., bacterium). In someexamples, the growth factor is EGF. In other examples, the mammal is apig and the EGF is porcine EGF.

The present disclosure further provides a method for increasing bindingof a microorganism (e.g., a bacterium) to intestinal mucosa (e.g., asmeasured by in vitro binding to a mucin preparation). Exemplary methodsinclude contacting the microorganism (e.g., bacterium) with an exogenousnucleic acid encoding a fusion protein, wherein the exogenous nucleicacid encoding a fusion protein comprises a sequence encoding a CmbApolypeptide; and expressing the exogenous nucleic acid encoding a fusionprotein in the microorganism (e.g., bacterium). In some examples, theexogenous nucleic acid encoding a fusion protein further comprises asequence encoding a mucin-binding polypeptide, such as a TFFpolypeptide. In some examples, expression of the exogenous nucleic acidencoding a fusion protein by the microorganism (e.g., bacterium)produces a fusion protein comprising the TFF polypeptide and the CmbApolypeptide.

In some examples according to any of the above described compositionsand methods, the microorganism is a non-pathogenic microorganism, e.g.,any microorganism safe for consumption by a mammalian subject. In someembodiments, the microorganism in the above compositions and methods isyeast. The yeast may be selected from Saccharomyces species, Hansenulaspecies, Kluyveromyces species, Schizzosaccharomyces species,Zygosaccharomyces species, Pichia species, Monascus species, Geothchumspecies, and Yarrowia species. In some examples, the yeast isSaccharomyces cerevisiae.

In other embodiments in the above compositions and methods, thenon-pathogenic microorganism is a non-pathogenic bacterium. In someexamples according to this embodiment, the non-pathogenic bacterium is aGram-positive bacterium. In other examples, the Gram-positive bacteriumis a lactic acid fermenting bacterium (LAB), e.g., is selected fromLactococcus species (e.g., Lactococcus lactis), Lactobacillus species,and Bifidobacterium species. In other examples, the non-pathogenicbacterium is a Streptococcus species or an Enterococcus species.Additional bacterial species are disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map of PthyA>>SSusp45-htff3-CmbA expression/thyAintegration vector pAGX2005. Abbreviations: thyA5′, 5′ flanking regionof the thymidine synthase A (thyA) gene of MG1363; PthyA, thyA promoterof MG1363; SSups45, gene that encodes for the secretion signal of MG1363protein usp45; hTFF3, gene that encodes for human trefoil factor 3 withoptimized codon usage for L. lactis; CmbA, gene that encodes forLactobacillus reuteri ATCC PTA6474 cell and mucus-binding protein A;thyA3′, 3′ flanking region of the thyA gene of MG1363; ORI, origin ofreplication of plasmid pORI19; Em, erythromycin resistance marker.

FIGS. 2A-B are graphs demonstrating enhanced binding of bacterial(Lactococcus lactis) cells to mucins and wherein mucin binding ismeasured using OD₄₀₅ (FIG. 2A) or crystal violet staining (FIG. 2B).pAGX1417: L. lactis strain LL108 that harbors an empty plasmid;pAGX1894: L. lactis strain LL108 that harbors a plasmid for theexpression of hTFF1-SpaX on the bacterial surface; pAGX2005: L. lactisstrain LL108 that harbors a plasmid for the expression of hTFF3-CmbA onthe bacterial surface. Mucin type II=mucin from porcine stomach, boundsialic acid, ˜1%; Mucin type III=mucin from porcine stomach, boundsialic acid (0.5-1.5%).

FIG. 3 demonstrates enhanced adherence of bacterial (Lactococcus lactis)cells to Caco-2 cells. pAGX1417: L. lactis strain LL108 that harbors anempty plasmid; pAGX1893: L. lactis strain LL108 that harbors a plasmidfor expressing CmbA on the bacterial surface; pAGX1894: L. lactis strainLL108 that harbors a plasmid for expressing hTFF1-spaX on the bacterialsurface; pAGX2005: L. lactis strain LL108 that harbors a plasmid forexpressing hTFF3-CmbA on the bacterial surface.

FIG. 4 illustrates an exemplary SSusp45-htff3 construct (amino acid andnucleic acid sequences: SEQ ID NO: 8 and SEQ ID NO: 9, respectively).

FIGS. 5A-D are collectively an illustration of an exemplarySSusp4S-hTFF3-CmbA construct (amino acid and nucleic acid sequences: SEQID NO: 10 and SEQ ID NO: 11, respectively).

FIG. 6 is a graph illustrating enhanced adherence of bacterial(Lactococcus lactis) cells to Caco-2 cells, wherein the cells eitherepisomally or constitutively express an exemplary cell adherencepolypeptide (CmbA) or an exemplary fusion protein (hTFF3-CmbA). %recovery=% of L. lactis cells that were recovered from the mucin coatedwell after washing compared to the total applied L. lactis cells, asdetermined by colony forming units count. pAGX1893: L. lactis strainLL108 that harbors a plasmid for expressing CmbA on the bacterialsurface; pAGX2005: L. lactis strain LL108 that harbors a plasmid forexpressing hTFF3-CmbA on the bacterial surface; sAGX0618: L. lactisstrain MG1363 constitutively expressing an exemplary therapeuticpolypeptide PAL (PgapB>>pal) and constitutively expressing CmbA on thebacterial surface (thyA⁻; PthyA>>cmbA); sAGX644: L. lactis strain MG1363constitutively expressing an exemplary therapeutic polypeptide PAL(PgapB>>pal) and constitutively expressing fusion protein hTFF3-CmbA onthe bacterial surface, wherein an exogenous nucleic acid encoding thefusion protein is transcriptionally regulated by an endogenous thyApromoter at the thyA locus (thyA⁻; PthyA>>SSusp45-htff3-mbA); sAGX660:L. lactis strain MG1363 constitutively expressing an exemplarytherapeutic polypeptide PAL (PgapB>>pal) and constitutively expressingthe fusion protein hTFF3-CmbA on the bacterial surface, wherein anexogenous nucleic acid encoding the fusion protein is transcriptionallyregulated by an hllA promoter at the thyA locus (thyA⁻;PhllA>>SSusp45-htff3-cmbA).

FIG. 7 is a graph illustrating enhanced binding of bacterial(Lactococcus lactis) cells to mucins, wherein the cells eitherepisomally or constitutively express an exemplary cell adherencepolypeptide (CmbA) or an exemplary fusion protein (hTFF3-CmbA), andwherein mucin binding is measured using OD₄₀₅ (FIG. 7A) or crystalviolet staining (FIG. 7B) as described herein, e.g., in Example 6.pAGX1893: L. lactis strain LL108 that harbors a plasmid for expressingCmbA on the bacterial surface; pAGX2005: L. lactis strain LL108 thatharbors a plasmid for expressing hTFF3-CmbA on the bacterial surface;sAGX0618: L. lactis strain MG1363 constitutively expressing an exemplarytherapeutic polypeptide PAL (PgapB>>pal) and constitutively expressingCmbA on the bacterial surface (thyA⁻; PthyA>>cmbA); sAGX644: L. lactisstrain MG1363 constitutively expressing an exemplary therapeuticpolypeptide PAL (PgapB>>pal) and constitutively expressing fusionprotein hTFF3-CmbA on the bacterial surface, wherein an exogenousnucleic acid encoding the fusion protein is transcriptionally regulatedby an endogenous thyA promoter at the thyA locus (thyA⁻;PthyA>>SSusp45-htff3-cmbA); sAGX660: L. lactis strain MG1363constitutively expressing an exemplary therapeutic polypeptide PAL(PgapB>>pal) and constitutively expressing the fusion protein htff3-CmbAon the bacterial surface, wherein an exogenous nucleic acid encoding thefusion protein is transcriptionally regulated by an hllA promoter at thethyA locus (thyA⁻; PhllA>>SSusp45-htff3-cmbA).

FIG. 8 is a graph illustrating production of an exemplary therapeuticpolypeptide (PAL) by bacterial cells either not expressing amucoadhesive polypeptide (sAGX0599), expressing the exemplary celladherence polypeptide CmbA (sAGX0618) on the bacterial surface, or theexemplary fusion protein hTFF3-CmbA on the bacterial surface (sAGX0644(thyA⁻; PthyA>>SSusp45-htff3-cmbA) and sAGX0660 (thyA⁻;PhllA>>SSusp45-htff3-cmbA)). PAL production in μg per 10⁹ cells wasfound comparable across PAL producing strains.

FIGS. 9A-D show that oral administration of L. lactis expressing PALlowers blood concentrations of phenylalanine. Negative CTR=no lactis,bolus only; Positive CTR=L. lactis strain NZ9000[pAGX1886], nisininduced PAL (AGX No 3151); L. lactis-PAL=sAGX0599 (AGX No 2947),secreting PAL; L. lactis-PAL+TFF3-CmbA=sAGX0645 mucoadherent strain (AGXNo 3290), secreting PAL and with surface TFF3-CmbA, which is cell andmucoadherent. FIGS. 9A-B show the blood levels of Phe and Tyr measuredover time following the administration of a bolus of radiolabeled Pheand FIGS. 9C-D show the corresponding AUC. The new constructs areassociated with lower levels of blood Phe. The final level of Phe waslowest in mice administered L. lactis expressing PAL and the TFF3-CmbAfusion protein.

DETAILED DESCRIPTION

The current disclosure provides microorganisms (e.g., lactic acidbacteria, such as Lactococcus lactis) exhibiting enhanced in vitrobinding to mucins and enhanced in vitro binding to cells, e.g.,adherence to Caco-2 cells. In some embodiments, such microorganisms havean increased GI transit time. For example, such microorganism mayadditionally express a therapeutic polypeptide. In some examples, uponoral administration of such bacteria to a mammalian subject, residencetime of the bacteria in the different parts of the GI-tract isincreased, and the subject is exposed to the therapeutic polypeptide inthe GI tract for a longer period of time. For example, once the bacteriaare released, e.g. from protective coated capsules in the duodenum,residence time of the bacteria in the jejunum and ileum is extended bysurface display/expression of muco and cell-adhesive proteins that bindor interact with the intestinal mucosa. Consequently, bacterial dosesmay be reduced, microorganisms with lower expression profiles becomeacceptable for administering therapeutically effective doses, smallerunit dosage forms can be developed, and regimens with less frequentadministration can be employed (e.g., increasing patient compliance).

For example, the present disclosure provides microorganisms containingan exogenous nucleic acid encoding a fusion protein of human trefoilfactor 3 (hTFF3) with cell and mucus binding protein A (CmbA) ofLactobacillus reuteri at the surface of Lactococcus lactis. An exemplaryfusion protein is composed of the secretion signal of Lactococcus lactisprotein usp45 (see. e.g., Van Asseldonk et al., Mol. Gen. Genet. 1993,240:428-434), fused to hTFF3 (see. e.g., Tomasetto et al.,Gastroenterology 2000, 118(1):70-80) and Lactobacillus reuteri CmbA(e.g., without its secretion signal). The SSusp45-hTFF3-CmbA fusionprotein is secreted by way of the usp45 secretion signal and thesecretion signal peptide is cleaved when the hTFF3-CmbA fusion proteinpasses the Lactococcus lactis cytoplasmic membrane. The external part ofCmbA can bind intestinal epithelial cells. By fusing hTFF3 to the CmbAprotein, an additional mucus binding unit is added. Expression andsurface display of hTFF3-CmbA enabled increased adherence to theintestinal mucosa and resulted in a slower GI-transit time of themodified Lactococcus lactis cells.

Definitions

As used in the specification and claims, the singular forms “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof. Similarly, use of “a compound” for treatmentor preparation of medicaments as described herein contemplates using oneor more compounds of this invention for such treatment or preparationunless the context clearly dictates otherwise.

The term “about” in relation to a reference numerical value, and itsgrammatical equivalents as used herein, can include the referencenumerical value itself and a range of values plus or minus 10% from thatreference numerical value. For example, the term “about 10” includes 10and any amounts from and including 9 to 11. In some cases, the term“about” in relation to a reference numerical value can also include arange of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%from that reference numerical value. In some embodiments, “about” inconnection with a number or range measured by a particular methodindicates that the given numerical value includes values determined bythe variability of that method.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

The percentage identity of polypeptide sequences can be calculated usingcommercially available algorithms which compare a reference sequencewith a query sequence. In some embodiments, polypeptides are 70%, atleast 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%,at least 90%, 92%, at least 92%, 95%, at least 95%, 97%, at least 97%,98%, at least 98%, 99%, or at least 99% or 100% identical to a referencepolypeptide, or a fragment thereof (e.g., as measured by BLASTP orCLUSTAL, or other alignment software) using default parameters.Similarly, nucleic acids can also be described with reference to astarting nucleic acid, e.g., they can be 50%, at least 50%, 60%, atleast 60%, 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%,at least 85%, 90%, at least 90%, 95%, at least 95%, 97%, at least 97%,98%, at least 98%, 99%, at least 99%, or 100% identical to a referencenucleic acid or a fragment thereof (e.g., as measured by BLASTN orCLUSTAL, or other alignment software using default parameters). When onemolecule is said to have a certain percentage of sequence identity witha larger molecule, it means that when the two molecules are optimallyaligned, the percentage of residues in the smaller molecule finds amatch residue in the larger molecule in accordance with the order bywhich the two molecules are optimally aligned, and the “%” (percent)identity is calculated in accord with the length of the smallermolecule.

It is to be understood that the expression of a foreign protein in abacterium typically requires modifications. These include modificationof the nucleic acid to remove of introns and other eukaryotic nucleicacid motifs that are not recognized by bacteria, and to optimize codonusage to the host.

Likewise, proteins are modified to remove motifs that are necessary forproper processing in the natural host but are not recognized by thebacterial host, such as secretion signals from eukaryotes or otherspecies of bacteria. Thus, when reference is made to a foreign proteinbeing expressed in bacteria, the person of ordinary skill wouldunderstand that to refer to the mature form. For example, human IL-10 istranslated in a human cell with a secretion leader sequence that is notpresent in the mature IL-10 secreted from the cell. The eukaryoticsecretion leader sequence is nonfunctional in bacteria. Accordingly, L.lactis further comprises nucleic acid expressing “IL-10”, it containsthe mature IL-10 protein.

As used herein, the term “expressing” a gene or polypeptide or“producing” a polypeptide (e.g., PAL, or an IL-2 polypeptide orT1D-specific antigen polypeptide) is meant to include “capable ofexpressing” and “capable of producing,” respectively. For example, amicroorganism, which contains an exogenous nucleic acid can, undersufficient conditions (e.g., sufficient hydration and/or in the presenceof nutrients), produce a polypeptide encoded by the exogenous nucleicacid). However, the microorganism may not always actively produce theencoded polypeptide. The microorganism (e.g., bacterium) may be dried(e.g., freeze-dried), and in that state can be considered dormant (i.e.,is not actively producing polypeptide). However, once the microorganismis subjected to sufficient conditions, e.g., is administered to asubject and is released (e.g., in the gastro-intestinal tract of thesubject) it may begin producing polypeptide. Thus, a microorganism“expressing” a gene or polypeptide or “producing” a polypeptide of thecurrent disclosure includes the microorganism in its “dormant” state.

As used herein, the term “constitutive” in the context of a promoter (orby extension relating to gene expression or secretion of a polypeptide)refers to a promoter that allows for continual transcription of itsassociated gene.

The term “chromosomally integrated” or “integrated into a chromosome” orany variation thereof means that a nucleic acid sequence (e.g., gene;open reading frame; exogenous nucleic acid encoding a polypeptide;promoter; expression cassette; and the like) is located on (integratedinto) a microbial (e.g., bacterial) chromosome, i.e., is not located onan episomal vector, such as a plasmid. In some embodiments, in which thenucleic acid sequence is chromosomally integrated, the polypeptideencoded by such chromosomally integrated nucleic acid is constitutivelyexpressed.

The terms “secretion leader sequence,” “secretion leader,” and“secretion signal sequence” are used interchangeably herein. The termsare used in accordance with their art recognized meaning, and generallyrefer to a nucleic acid sequence, which encodes a “signal peptide” or“secretion signal peptide” causes a polypeptide being expressed by amicroorganism and comprising the signal peptide to be secreted by themicroorganism, i.e., causes the polypeptide to leave the intracellularspace, e.g., be secreted into the surrounding medium, or be anchored inthe cell wall with at least a portion of the polypeptide be exposed tothe surrounding medium, e.g. on the surface of the microorganism.

Therapeutic Polypeptide

The term “therapeutic polypeptide” includes any polypeptide that has atherapeutic, prophylactic, or other biological activity (e.g., in amammalian subject), or has the potential for eliciting a biologicalactivity. Examples include known biologics (approved andinvestigational), and any signal polypeptides, such as hormones andcytokines, and their receptors, agonists and antagonists. A “therapeuticpolypeptide” may be modified from a corresponding wild-type polypeptide.In some examples, the therapeutic polypeptide is a cytokine, e.g., aninterleukin (IL), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21 or IL-22.

In other examples, the therapeutic polypeptide is an antigen. In someexamples according to this embodiment, the antigen is an “auto-antigen”or self-antigen. The terms “self-antigen” or “auto-antigen” are usedinterchangeably herein. The terms are used herein in accordance with theart recognized meaning of self-antigen or auto-antigen, and generallyrefer to a polypeptide/protein originating from within a subjects ownbody (produced by the subject's own body), wherein the antigen isrecognized by the subject's own immune system, and typically producesantibodies against such antigen. Autoimmune diseases are generallyassociated with certain disease-specific self-antigens. For example, inT1D a subject's immune system may produce antibodies against at leastone antigen associated with the beta-cell destruction process. In someexamples, the auto-antigen is a T1D-specific antigen. ExemplaryT1D-specific antigens include proinsulin (PINS), glutamic aciddecarboxylase (GAD65), insulinoma-associated protein 2 (IA-2),islet-specific glucose-6-phosphatase catalytic subunit-related protein(IGRP), zinc transporter 8 (ZnT8), and any combinations thereof.Clinical T1D may further be associated with additional candidate targetmolecules expressed by beta-cells such as chromogranin A, (prepro) isletamyloid polypeptide (ppIAPP), peripherin, and citrullinatedglucose-regulated protein (GRP), and any combinations thereof. Exemplaryamino acid sequences and nucleic acid sequences for the aboveT1D-specific antigens are disclosed, e.g., in provisional patentapplication 62/350,472 (filed Jun. 15, 2016), the disclosure of which isincorporated herein by reference in its entirety. In some examples, theT1D-specific antigen is PINS, such as wild-type human PINS. See, e.g.,CDS contained in accession number NM_000207.2, or a sequence that is atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% sequence identity with suchwild-type human PINS. Additional exemplary PINS nucleotide sequences arerepresented by the coding sequences of NCBI accession numbers AY899304(complete CDS, alternatively spliced); NM_000207 (transcript variant 1);NM_001185097 (transcript variant 2); NM_001185098 (transcript variant3); NM_001291897 (transcript variant 4), and partial sequences thereof.Exemplary PINS amino acid sequences include those encoded by any one ofthe above PINS nucleic acid sequences.

In other examples, the antigen is an allergen, such as a tree pollenallergen, a weed pollen allergen, a grass pollen allergen, a foodallergen, a dust-mite allergen, a mold allergen, an animal danderallergen, or a combination thereof. In some examples, the allergen is aweed pollen allergen, e.g., a ragweed pollen allergen. In otherexamples, the allergen is a tree pollen allergen, such as a birch pollenallergen or a Japanese cedar pollen allergen. In yet other examples, theallergen is a food allergen, such as a peanut allergen, a milk allergen,an egg allergen, a gluten allergen (gliadin epitope), or a combinationthereof. In other examples the therapeutic polypeptide is an antigen andan interleukin, such as IL-2, IL-10, or IL-22.

In further examples, the therapeutic polypeptide is an antibody or afragment thereof. For example, the antibody is a single-domain antibodyor a nanobody. Exemplary antibodies include cytokine neutralizingantibodies such as antibodies to IL-4, antibodies to IL-5, antibodies toIL-7, antibodies to IL-13, antibodies to IL-15, as well as anti TNFαantibodies, antibodies to immunoglobulin E (IgE), anti-P40, and anyfragments thereof.

In yet other examples, the therapeutic polypeptide is an enzyme or afragment (e.g., functional fragment) thereof, e.g., a phenylalanineammonia lyase (PAL), an amino acid decarboxylase, or a combinationthereof. In one example, the therapeutic polypeptide is PAL, or afunctional fragment thereof. Exemplary PAL sequences useful for thisembodiment are disclosed, e.g., in International Patent ApplicationPublication WO 2014/066945, the disclosure of which is incorporatedherein by reference in its entirety. PAL metabolizes phenylalanine andthereby can reduce the level of Phe absorbed from the gut into theblood, and therefore can be used to treat phenylketonuria. Other enzymesmay also be used to degrade Phe, such as the aromatic amino aciddecarboxylases, such as phenylalanine decarboxylases. In someembodiments, the subject is administered bacteria that expresses andsecretes PAL and second phenylalanine degrading enzyme. In anotherembodiment the subject is administered a bacteria that expresses andsecretes PAL, and another bacteria that expresses and secretes a secondphenylalanine degrading enzyme.

In a further example, a bacteria is engineered to enhance Phe uptake andutilization within the cell. Administration of such bacteria can furtherreduce the amount of Phe absorbed by the patient.

In further examples, the therapeutic polypeptide is a glucagon-likepeptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), glucagon, exendin-4,or any combination thereof. In other examples, the therapeuticpolypeptide is a growth factor, such as an epidermal growth factor(EGF), e.g., human EGF or porcine EGF. In yet other examples, thetherapeutic polypeptide is a TFF, such as TFF1, TFF2, TFF3, or acombination thereof.

In some examples, the therapeutic polypeptide is an immuno-modulatingcompound. The terms “immuno-modulating compound” or immuno-modulator”are used herein in accordance with their art-recognized meaning. Theimmuno-modulating compound can be any immuno-modulating compound knownto a person skilled in the art. In some embodiments, theimmuno-modulating compound is a tolerance inducing compound. Toleranceinduction can be obtained, e.g., by inducing regulatory T-cells, or inan indirect way, e.g., by activation of immature dendritic cells totolerizing dendritic cells and/or inhibiting Th2 immune responseinducing expression of “co-stimulation” factors on mature dendriticcells. Immuno-modulating and immuno-suppressing compounds are known tothe person skilled in the art and include, but are not limited to,bacterial metabolites such as spergualin, fungal and streptomycalmetabolites such as tacrolimus or ciclosporin, immuno-suppressingcytokines such as IL-4, IL-10, IFNα, TGFβ (as selective adjuvant forregulatory T-cells) FIt3L, TSLP and Rank-L (as selective tolerogenic DCinducers), antibodies and/or antagonist (e.g., antibodies) such asanti-CD40L, anti-CD25, anti-CD20, anti-IgE, anti-CD3, and proteins,peptides or fusion proteins such as the CTL-41 g or CTLA-4 agonistfusion protein. In some embodiments, the immuno-modulating compound isan immuno-suppressing compound. In other embodiments, theimmuno-suppressing compound is an immuno-suppressing cytokine orantibody. In other embodiments, the immuno-suppressing cytokine is atolerance-enhancing cytokine or antibody. It will be appreciated by theperson skilled in the art that the term “immuno-modulating compound”also includes functional homologues thereof. A functional homologue is amolecule having essentially the same or similar function for theintended purposes, but can differ structurally. In some examples, theimmuno-modulating compound is an anti-CD3 antibody, or a functionalhomologue thereof.

A microorganism of the present disclosure may express more than one, orat least one therapeutic polypeptide. The therapeutic polypeptide may bea combination of any of the above recited therapeutic polypeptides.

Diseases

The microorganisms (e.g., bacteria), compositions and methods of thepresent disclosure can be used to treat or prevent any disease, e.g.,those which can be treated by a bioactive polypeptide that is active atthe site of the mucosa, e.g., gastro-intestinal mucosa. Exemplarydiseases that can be treated or prevented using the methods of thepresent disclosure include autoimmune disease, allergies, nutritional ormetabolic diseases, gastro-intestinal diseases, and genetic disorders,or any combinations thereof.

The term “nutritional disease” includes any disease that is associatedwith an insufficiency to process food or nutrients, and may result,e.g., in malnutrition, low weight, or other secondary conditions (suchas bloating). A “nutritional disease” may be associated withinsufficient production of certain enzymes that process food or foodcomponents, such as lipids and carbohydrates (such as lipases,proteases, or sugar degrading enzymes). The term “nutritional disease”includes any metabolic process in an organism that can be enhanced evenif no defined condition or disease is present (“metabolic enhancement”),e.g., certain farm animals, such as pigs, cows, birds, or sheep can betreated to grow faster or accumulate higher weights. In some examples,the “nutritional disease” is an intolerance to certain foods or foodcomponents based on insufficient or abnormal metabolism of such food orfood component, such as lactose intolerance. The term “nutritionaldisease” is related to “metabolic disease” or “metabolic disorder” usedinterchangeably herein. The term “metabolic disease” is used herein inaccordance with its art-recognized meaning, and generally refers to anycondition, in which abnormal chemical reactions in the body alter anormal metabolic process. In some examples, the metabolic disease iscaused by a genetic defect, and may be inherited. Examples of metabolicdisorder include acid-base imbalances, metabolic brain diseases, calciummetabolism disorders, DNA repair-deficiency disorders, glucosemetabolism disorders, hyperlactatemia, iron metabolism disorders, andlipid metabolism disorders.

Other examples of nutritional or metabolic diseases include glucoseand/or galactose malabsorption, Lesch-Nyhan syndrome, Menkes syndrome,obesity, pancreatic cancer, Prader-Willi syndrome, porphyria, Refsumdisease, Tangier disease, Wilson's disease, Hurler syndrome (e.g.,characterized by abnormal bone structure and developmental delay),Niemann-Pick disease (e.g., in which babies develop liver enlargement,difficulty feeding, and nerve damage), Tay-Sachs disease (e.g.,characterized by progressive weakness in a young child, progressing tosevere nerve damage), Gaucher disease (e.g., characterized by bone pain,enlarged liver, and low platelet counts); Fabry disease (e.g.,characterized by pain in the extremities in childhood, with kidney andheart disease and strokes in adulthood), Krabbe disease (e.g.,characterized by progressive nerve damage, developmental delay in youngchildren); galactosemia (e.g., characterized by impaired breakdown ofthe sugar galactose, can lead to jaundice, vomiting, and liverenlargement after breast or formula feeding by a newborn); maple syrupurine disease (e.g., characterized by deficiency of the enzyme BCKD,causes buildup of amino acids in the body); phenylketonuria (PKU),glycogen storage diseases (e.g., characterized by low blood sugarlevels, muscle pain, and weakness); mitochondrial disorders, Friedreichataxia (e.g., characterized by problems related to the protein frataxin,which may cause nerve damage, heart problems, inability to walk), andperoxisomal disorders (e.g., characterized by poor enzyme functioninside peroxisomes, which may lead to buildup of toxic metabolites).Exemplary peroxisomal disorders include, e.g., Zellweger syndrome (e.g.,characterized by abnormal facial features, enlarged liver, and nervedamage in infants), and adrenoleukodystrophy (e.g., characterized bysymptoms of nerve damage in childhood or early adulthood). Othernutritional or metabolic disorders include metal metabolism disorders(e.g., characterized by protein malfunction and toxic accumulation ofmetal in the body). Examples include, e.g., Wilson disease (e.g.,characterized by accumulation of toxic copper levels in the liver,brain, and other organs), and hemochromatosis (e.g., hereditaryhemochromatosis), e.g., in which the intestines absorb excessive iron,which builds up in the liver, pancreas, joints, and heart, causingdamage. Further examples of nutritional or metabolic disorders includeorganic acidemias (such as methylmalonic acidemia and propionicacademia), urea cycle disorders (such as ornithine transcarbamylasedeficiency and citrullinemia). In some example, the nutritional ormetabolic disease is phenylketonuria (PKU). In other examples, thenutritional or metabolic disease is a metabolic disorder related toenergy dysregulation (e.g., nonalcoholic steatohepatitis).

In some examples, the disease is an autoimmune disease. Exemplaryautoimmune diseases include myocarditis, postmyocardial infarctionsyndrome, postpericardiotomy syndrome, subacute bacterial endocarditis(SBE), anti-glomerular basement membrane nephritis, interstitialcystitis, lupus nephritis, autoimmune hepatitis, primary biliarycirrhosis (PBC), primary sclerosing cholangitis, antisynthetasesyndrome, alopecia areata, autoimmune angioedema, autoimmuneprogesterone dermatitis, autoimmune urticarial, bullous pemphigoid,cicatricial pemphigoid, dermatitis herpetiformis, discoid lupuserythematosus, epidermolysis bullosa acquisita, erythema nodosum,gestational pemphigoid, hidradenitis suppurativa, lichen planus, lichensclerosus, linear IgA disease (LAD), morphea, pemphigus vulgaris,pityriasis lichenoides et varioliformis acuta, Mucha-Habermann disease,psoriasis, systemic scleroderma, vitiligo, Addison's disease, autoimmunepolyendocrine syndrome (APS) type 1, autoimmune polyendocrine syndrome(APS) type 2, autoimmune polyendocrine syndrome (APS) type 3, autoimmunepancreatitis (AIP), diabetes mellitus type 1, autoimmune thyroiditis,Ord's thyroiditis, Graves' disease, autoimmune oophoritis,endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmuneenteropathy, Coeliac disease, Crohn's disease, microscopic colitis,ulcerative colitis, antiphospholipid syndrome (APS, APLS), aplasticanemia, autoimmune hemolytic anemia, autoimmune lymphoproliferativesyndrome, autoimmune neutropenia, autoimmune thrombocytopenic purpura,cold agglutinin disease, essential mixed cryoglobulinemia, Evanssyndrome, paroxysmal nocturnal, hemoglobinuria, pernicious anemia, purered cell aplasia, thrombocytopenia, adiposis dolorosa, adult-onsetStill's disease, ankylosing spondylitis, CREST syndrome, drug-inducedlupus, enthesitis-related arthritis, eosinophilic fasciitis, Feltysyndrome, IgG4-related disease, juvenile arthritis, Lyme disease(chronic), mixed connective tissue disease (MCTD), palindromicrheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, psoriaticarthritis, reactive arthritis, relapsing polychondritis, retroperitonealfibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schnitzlersyndrome, systemic lupus erythematosus (SLE), undifferentiatedconnective tissue disease (UCTD), dermatomyositis, fibromyalgia,inclusion body myositis, myositis, myasthenia gravis, neuromyotonia,paraneoplastic cerebellar degeneration, polymyositis, acute disseminatedencephalomyelitis (ADEM), acute motor axonal neuropathy,anti-N-methyl-D-aspartate (anti-NMDA) receptor encephalitis, baloconcentric sclerosis, Bickerstaff's encephalitis, chronic inflammatorydemyelinating polyneuropathy, Guillain-Barré syndrome, Hashimoto'sencephalopathy, idiopathic inflammatory demyelinating diseases,Lambert-Eaton myasthenic syndrome, multiple sclerosis (MS), pattern II,Oshtoran Syndrome, pediatric autoimmune neuropsychiatric disorderassociated with streptococcus (PANDAS), progressive inflammatoryneuropathy, restless leg syndrome, stiff person syndrome, Sydenhamchorea, transverse myelitis, autoimmune retinopathy, autoimmune uveitis,Cogan syndrome, Graves ophthalmopathy, intermediate uveitis, ligneousconjunctivitis, Mooren's ulcer, neuromyelitis optica, opsoclonusmyoclonus syndrome, optic neuritis, scleritis, Susac's syndrome,sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune inner eardisease (AIED), Méniére's disease, Behçet's disease, eosinophilicgranulomatosis with polyangiitis (EGPA), giant cell arteritis,granulomatosis with polyangiitis (GPA), IgA vasculitis (IgAV),Kawasaki's disease, leukocytoclastic vasculitis, lupus vasculitis,rheumatoid vasculitis, microscopic polyangiitis (MPA), polyarteritisnodosa (PAN), polymyalgia rheumatic, urticarial vasculitis, vasculitis,and primary immune deficiency. In some examples, the autoimmune diseaseis type-1 diabetes (T1D).

In other examples, the disease is a gastro-intestinal disease, such asshort bowel syndrome, celiac disease, or inflammatory bowel disease(IBD), e.g., Crohn's disease or ulcerative colitis.

In some examples, the disease is an inflammatory disease (e.g., Th2and/or IgE driven inflammation). Exemplary inflammatory diseases includeacne vulgaris, asthma, autoinflammatory diseases, chronic prostatitis,glomerulonephritis, hypersensitivities, inflammatory bowel diseases(IBD), pelvic inflammatory disease, reperfusion injury, rheumatic fever,rheumatoid arthritis, sarcoidosis, transplant rejection,graft-versus-host disease, vasculitis, hydradenitis suppurativa,diverticulitis, interstitial cystitis. Examples of autoinflammatorydiseases include familial Mediterranean fever (FMF),hyperimmunoglobulinemia D with recurrent fever (HIDS), mevalonicaciduria, mevalonate kinase deficiency, TNF receptor associated periodicsyndrome (TRAPS), Muckle-Wells syndrome (urticaria deafnessamyloidosis), familial cold urticarial, neonatal onset multisysteminflammatory disease (NOMID), periodic fever, aphthous stomatitis,pharyngitis and adenitis (PFAPA syndrome), Blau syndrome, pyogenicsterile arthritis, pyoderma gangrenosum, acne (PAPA), deficiency of theinterleukin-1-receptor antagonist (DIRA).

In further examples, the disease is growth retardation. In otherexamples, the disease is type-2 diabetes (T2D), obesity, or pain (e.g.,neuropathic pain).

In other examples, the disease is an allergy, e.g., an allergy to anallergen selected from a tree pollen allergen, a weed pollen allergen, agrass pollen allergen, a food allergen, a dust-mite allergen, a moldallergen, an animal dander allergen, or a combination thereof. In someexamples, the disease is an allergy to a weed pollen allergen, e.g., aragweed pollen allergen. In other examples, the disease is an allergy toa tree pollen allergen, such as a birch pollen allergen or a Japanesecedar pollen allergen. In yet other examples, the disease is an allergyto a food allergen, such as a peanut allergen, a milk allergen, an eggallergen, a gluten allergen (gliadin epitope), or a combination thereof.

Phenylketonuria

In some examples, the present disclosure provides methods for thetreatment of phenylketonuria (PKU). The term “phenylketonuria” is usedherein in accordance with its art-recognized meaning. Phenylketonuria(PKU) is one of the most prevalent disorders of amino acid metabolism.Genetic defects (deficiency of the enzyme PAH) result in high levels ofblood phenylalanine (Phe), which can lead to severe mental retardationif not recognized, and treated early in life. Even with dietarycompliance, PKU patients risk cognitive impairment from adolescenceonward.

Promoter

By “promoter” is meant generally a region on a nucleic acid molecule,for example DNA molecule, to which an RNA polymerase binds and initiatestranscription. A promoter is for example, positioned upstream, i.e., 5′,of the sequence the transcription of which it controls. The skilledperson will appreciate that the promoter may be associated withadditional native regulatory sequences or regions, e.g. operators. Theprecise nature of the regulatory regions needed for expression may varyfrom organism to organism, but shall in general include a promoterregion which, in prokaryotes, contains both the promoter (which directsthe initiation of RNA transcription) as well as the DNA sequences which,when transcribed into RNA, will signal the initiation of proteinsynthesis. Such regions will normally include those 5′-non-codingsequences involved with initiation of transcription and translation,such as the Pribnow-box (cf. TATA-box), Shine-Dalgarno sequence, and thelike.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences. For example, a promoter is said to be operably linked to agene, open reading frame or coding sequence, if the linkage orconnection allows or effects transcription of said gene. In a furtherexample, a 5′ and a 3′ gene, cistron, open reading frame or codingsequence are said to be operably linked in a polycistronic expressionunit, if the linkage or connection allows or effects translation of atleast the 3′ gene. For example, DNA sequences, such as, e.g., a promoterand an open reading frame, are said to be operably linked if the natureof the linkage between the sequences does not (1) result in theintroduction of a frameshift mutation, (2) interfere with the ability ofthe promoter to direct the transcription of the open reading frame, or25 (3) interfere with the ability of the open reading frame to betranscribed by the promoter region sequence.

Expression Cassette

The term “expression cassette” or “expression unit” is used inaccordance with its generally accepted meaning in the art, and refers toa nucleic acid containing one or more genes and sequences controllingthe expression of the one or more genes. Exemplary expression cassettescontain at least one promoter sequence and at least one open readingframe.

Polycistronic Expression Cassette

The terms “polycistronic expression cassette,” “polycistronic expressionunit,” or “polycistronic expression system” are used hereininterchangeably and in accordance with their generally accepted meaningin the art. They refer to a nucleic acid sequence wherein the expressionof two or more genes is regulated by common regulatory mechanisms, suchas promoters, operators, and the like. The term polycistronic expressionunit as used herein is synonymous with multicistronic expression unit.Examples of polycistronic expression units are without limitationbicistronic, tricistronic, tetracistronic expression units. Any mRNAcomprising two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, or more, openreading frames or coding regions encoding individual expression productssuch as proteins, polypeptides and/or peptides is encompassed within theterm polycistronic. A polycistronic expression cassette includes atleast one promoter, and at least two open reading frames controlled bythe promoter, wherein an intergenic region is optionally placed betweenthe two open reading frames.

In some example, the “polycistronic expression cassette” includes one ormore endogenous genes and one or more exogenous genes that aretranscriptionally controlled by a promoter which is endogenous to themicroorganism (e.g., LAB). In another embodiment, the polycistronicexpression unit or system as described herein is transcriptionallycontrolled by a promoter which is exogenous to the microorganism (e.g.,LAB). In a further embodiment, the translationally or transcriptionallycoupled one or more endogenous genes and one or more exogenous genes asdescribed herein are transcriptionally controlled by the native promoterof (one of) said one or more endogenous genes. In another embodiment,the polycistronic expression unit is transcriptionally controlled by thenative promoter of (one of) said one or more endogenous genes comprisedin said polycistronic expression unit. In another embodiment, thepolycistronic expression unit is operably linked to a Gram-positiveendogenous promoter. In an exemplary embodiment, the promoter may bepositioned upstream of, i.e., 5′ of the open reading frame(s) to whichit is operably linked. In a further embodiment, the promoter is thenative promoter of the 5′ most, i.e., most upstream, endogenous gene inthe polycistronic expression unit. Accordingly, in some examples, thepolycistronic expression unit contains an endogenous gene and one ormore exogenous genes transcriptionally coupled to the 3′ end of said oneor more endogenous gene, for example wherein said one or more exogenousgene(s) is (are) the most 3′ gene(s) of the polycistronic expressionunit.

As used herein, the term “translationally coupled” is synonymous with“translationally linked” or “translationally connected”. These terms inessence relate to polycistronic expression cassettes or units. Two ormore genes, open reading frames or coding sequences are said to betranslationally coupled when common regulatory element(s) such as inparticular a common promoter effects the transcription of said two ormore genes as one mRNA encoding said two or more genes, open readingframes or coding sequences, which can be subsequently translated intotwo or more individual polypeptide sequences. The skilled person willappreciate that bacterial operons are naturally occurring polycistronicexpression systems or units in which two or more genes aretranslationally or transcriptionally coupled.

Intergenic Region

As used herein, the term “intergenic region” is synonymous with“intergenic linker” or “intergenic spacer”. An intergenic region isdefined as a polynucleic acid sequence between adjacent (i.e., locatedon the same polynucleic acid sequence) genes, open reading frames,cistrons or coding sequences. By extension, the intergenic region caninclude the stop codon of the 5′ gene and/or the start codon of the 3′gene which are linked by said intergenic region. As defined herein, theterm intergenic region specifically relates to intergenic regionsbetween adjacent genes in a polycistronic expression unit. For example,an intergenic region as defined herein can be found between adjacentgenes in an operon. Accordingly, in an embodiment, the intergenic regionas defined herein is an operon intergenic region.

In some examples, the intergenic region, linker or spacer is selectedfrom intergenic regions preceding, i.e., 5′ to, more particularlyimmediately 5′ to, rplW, rpl P, rpmD, rplB, rpsG, rpsE or rplN of aGram-positive bacterium. In some embodiments, the Gram-positivebacterium is a lactic acid bacterium, for example a Lactococcus species,e.g., Lactococcus lactis, and any subspecies or strain thereof. In anembodiment, said intergenic region encompasses the start codon of rplW,rpl P, rpmD, rplB, rpsG, rpsE or rplN and/or the stop codon of thepreceding, i.e. 5′, gene. In some embodiments, the invention relates toa Gram-positive bacterium or a recombinant nucleic acid as describedherein, wherein the endogenous gene and the one or more exogenous genesare transcriptionally coupled by intergenic region or regions active inthe Gram-positive bacterium, for example wherein the intergenic regionor regions is endogenous to said Gram-positive bacterium, for example,wherein the endogenous intergenic region is selected from intergenicregions preceding rplW, rpl P, rpmD, rplB, rpsG, rpsE or rplN rplM,rplE, and rplF.

The skilled person will appreciate that if the intergenic regionencompasses a 5′ stop codon and/or a 3′ start codon, these respectivecodons in some cases are not present in the genes which are linked bysaid intergenic regions, in order to avoid double start and/or stopcodons, which may affect correct translation initiation and/ortermination. Methods for identifying intergenic regions are known in theart. By means of further guidance, intergenic regions can for instancebe identified based on prediction of operons, and associated promotersand open reading frames, for which software is known and available inthe art. Exemplary intergenic regions are described in internationalpatent application publication WO2012/164083, the disclosure of which isincorporated herein by reference in its entirety.

Subject

A “subject” is an organism, which may benefit from being administered acomposition of the present disclosure, e.g., according to methods of thepresent disclosure. The subject may be a mammal (“mammalian subject”).Exemplary mammalian subjects include humans, farm animals (such as cows,pigs, horses, sheep, goats), pets (such as a dogs, cats, and rabbits),and other animals, such as mice, rats, and primates. In some examples,the mammalian subject is a human patient.

Mucosa

The term “mucosa” or “mucous membrane” is used herein in accordance withits art recognized meaning. The “mucosa” can be any mucosa found in thebody, such as oral mucosa, rectal mucosa, gastric mucosa, intestinalmucosa, urethral mucosa, vaginal mucosa, ocular mucosa, buccal mucosa,bronchial or pulmonary mucosa, and nasal or olfactory mucosa. Mucosa mayalso refer to surface mucosa, e.g., those found in fish and amphibia.

The term “mucosal delivery” as used herein is used in accordance withits art recognized meaning, i.e., delivery to the mucosa, e.g., viacontacting a composition of the present disclosure with a mucosa. Oralmucosal delivery includes buccal, sublingual and gingival routes ofdelivery. Accordingly, in some embodiments, “mucosal delivery” includesgastric delivery, intestinal delivery, rectal delivery, buccal delivery,pulmonary delivery, ocular delivery, nasal delivery, vaginal deliveryand oral delivery. The person of ordinary skill will understand thatoral delivery can affect delivery to distal portions of thegastrointestinal tract.

The term “mucosal tolerance” refers to the inhibition of specific immuneresponsiveness to an antigen in a mammalian subject (e.g., a humanpatient), after the subject has been exposed to the antigen via themucosal route. In some cases the mucosal tolerance is systemictolerance. Low dose oral tolerance is oral tolerance induced by lowdoses of antigens, and is characterized by active immune suppression,mediated by cyclophosphamide sensitive regulatory T-cells that cantransfer tolerance to naive hosts. High dose oral tolerance is oraltolerance induced by high doses of antigens, is insensitive tocyclophosphamide treatment, and proceeds to induction of T cellhyporesponsiveness via anergy and/or deletion of antigen specificT-cells. The difference in sensitivity to cyclophosphamide can be usedto make a distinction between low dose and high dose tolerance (Strobelet al., 1983). In some cases, the oral tolerance is low dose oraltolerance as described by Mayer and Shao (2004).

Mucin

The term “mucin” is used herein in accordance with its art-recognizedmeaning. Mucins are a family of high molecular weight, glycosylatedproteins (glycoconjugates) produced by epithelial tissues in humans andanimals. Mucins have the ability to form gels, and are a key componentof gel-like secretions. Some mucins are membrane-bound due to thepresence of a hydrophobic membrane-spanning domain. Most mucins aresecreted as principal components of mucus by mucous membranes or aresecreted to become a component of saliva. Mucin genes include MUC1,MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC12, MUC13,MUC15, MUC16, MUC17. MUC9, MUC20, and MUC21, MUC2 is secreted mostly inthe intestine but also in the airway. Mature mucins are composed of twodistinct regions: amino- and carboxy-terminal regions are lightlyglycosylated, but rich in cysteines. The cysteine residues participatein establishing disulfide linkages within and among mucin monomers. Alarge central region formed of multiple tandem repeats of 10 to 80residue sequences in which up to half of the amino acids are serine orthreonine. This area becomes saturated with mostly O-linkedoligosaccharides. Overexpression of mucin proteins (e.g., MUC1) isassociated with many types of cancer. In the context of this disclosure“mucin” may also mean “mucin preparation” or “mucous or otherpreparation containing mucins.”

Treating

The terms “treatment”, “treating”, and the like, as used herein meansameliorating or alleviating characteristic symptoms or manifestations ofa disease or condition, e.g., PKU or T1D. For example, treatment of T1Dcan result in the restoration or induction of antigen-specific immunetolerance in the subject. In other examples, treatment means arrestingautoimmune diabetes, or reversing autoimmune diabetes. As used hereinthese terms also encompass, preventing or delaying the onset of adisease or condition or of symptoms associated with a disease orcondition, including reducing the severity of a disease or condition orsymptoms associated therewith prior to affliction with said disease orcondition. Such prevention or reduction prior to affliction refers toadministration of the microorganism (e.g., bacterium) or composition ofthe present disclosure to a patient that is not at the time ofadministration afflicted with the disease or condition. “Preventing”also encompasses preventing the recurrence or relapse-prevention of adisease or condition or of symptoms associated therewith, for instanceafter a period of improvement.

Treatment of a subject “in need thereof” conveys that the subject has adiseases or condition, and the therapeutic method of the invention isperformed with the intentional purpose of treating the specific diseaseor condition.

Therapeutically Effective Amount

As used herein, the term “therapeutically effective amount” refers to anamount of a non-pathogenic microorganism or a composition of the presentdisclosure that will elicit a desired therapeutic effect or responsewhen administered according to the desired treatment regimen. In someexamples, the compounds or compositions are provided in a unit dosageform, for example a tablet or capsule, which contains an amount of theactive component equivalent with the therapeutically effective amountwhen administered once, or multiple times per day.

A person of ordinary skill in the art will appreciate that atherapeutically effective amount of a recombinant microorganism, whichis required to achieve a desired therapeutic effect (e.g., for theeffective treatment of T1D), will vary, e.g., depending on the nature ofthe polypeptide expressed by the microorganism, the route ofadministration, and the age, weight, and other characteristics of therecipient.

The amount of secreted polypeptide can be determined based on cfu,determined by state of the art methods such as Q-PCR, or by using ELISA.For example, a particular microorganism may secrete at least about 1 ngto about 1 μg of active polypeptide per 10⁹ cfu. Based thereon, theskilled person can calculate the range of antigen polypeptide secretedat other cfu doses.

Therapeutically effective amounts may be administered in connection withany dosing regimen as described herein. The daily dose of activepolypeptide may be administered in 1, 2, 3, 4, 5, or 6 portionsthroughout the day. Further the daily doses may be administered for anynumber of days, with any number of rest periods between administrationperiods. For example, a dose of the active agent (e.g. interleukin) offrom about 0.01 to about 3.0 M IU/day/subject may be administered everyother day for a total of 6 weeks. In other examples. PAL is administeredat doses ranging from 0.1 to 1000 mg per day, such as doses of 1-100 mgat each meal.

T1D-Specific Antigen

In some embodiments, in any of the above compositions and methods, theT1D-specific antigen is selected from known auto-antigens implemented inT1D, and include proinsulin (PINS); insulin (INS); glutamic aciddecarboxylase (GAD) (e.g., GAD65, GAD67, or GAD2); insulinoma-associatedprotein 2 (islet antigen-2; IA-2) (also referred to as protein tyrosinephosphatase, receptor type, N (PTPRN), tyrosine phosphatase-likeprotein, or ICA512), (see, e.g., Long et al., Diabetes 2013, 62 (6),2067-2071); islet-specific glucose-6-phosphatase catalyticsubunit-related protein (IGRP), zinc transporter 8 (ZnT8), chromograninA, (prepro) islet amyloid polypeptide (ppIAPP), peripherin,citrullinated glucose-regulated protein (e.g., GRP78); see, e.g., Rondaset al., Diabetes 2015; 64(2):573-586; and Ye et al., Diabetes 2010,59(1):6-16), and combinations of two or more of these antigens. In otherembodiments, in the above compositions and methods, the T1D-specificantigen is PINS, GAD65, or IA-2. In other embodiments, in the abovecompositions and methods, the T1D-specific antigen is PINS. In variousembodiments, the T1D-specific antigen is encoded by a variant nucleicacid sequence shorter than a full length (e.g., wild-type) gene, as such“trimmed” versions are often more efficiently expressed and/or secretedby the microorganisms used (e.g., Lactococcus lactis). While secretionis more efficient, many “trimmed” versions retain all (or a substantialportion) of their biological activity, e.g., retain sufficient Tregstimulating and/or tolerance-inducing capacities.

Microorganism

In some examples according to any of the embodiments presented herein,the microorganism is a non-pathogenic microorganism, e.g., anon-pathogenic and non-invasive bacterium. In other embodiments, themicroorganism is a non-pathogenic and non-invasive yeast.

In some embodiments, the microorganism is a yeast strain selected fromthe group consisting of Saccharomyces sp., Hansenula sp., Kluyveromycessp., Schizzosaccharomyces sp., Zygosaccharomyces sp., Pichia sp.,Monascus sp., Geothchum sp. and Yarrowia sp. In some embodiments, theyeast is Saccharomyces cerevisiae. In other embodiments, the S.cerevisiae is of the subspecies boulardii. In one embodiment of thepresent invention, the recombinant yeast host-vector system is abiologically contained system. Biological containment is known to theperson skilled in the art and can be realized by the introduction of anauxotrophic mutation, e.g., a suicidal auxotrophic mutation such as thethyA mutation, or its equivalents. Alternatively, the biologicalcontainment can be realized at the level of the plasmid carrying thegene encoding the polypeptide, such as, for example, by using anunstable episomal construct, which is lost after a few generations.Several levels of containment, such as plasmid instability andauxotrophy, can be combined to ensure a high level of containment, ifdesired.

In other embodiments of the present invention, the microorganism is abacterium, such as a non-pathogenic bacterium, e.g., a food gradebacterial strain. In some examples, the non-pathogenic bacterium is aGram-positive bacterium, e.g., a Gram-positive food-grade bacterialstrain. In some embodiments, the Gram-positive food-grade bacterialstrain is a lactic acid fermenting bacterial strain (i.e., a lactic acidbacterium (LAB)) or a Bifidobacterium.

In some embodiments, the lactic acid fermenting bacterial strain is aLactococcus, Lactobacillus or Bifidobacterium species. As used herein,Lactococcus or Lactobacillus is not limited to a particular species orsubspecies, but meant to include any of the Lactococcus or Lactobacillusspecies or subspecies. Exemplary Lactococcus species include Lactococcusgarvieae, Lactococcus lactis, Lactococcus piscium, Lactococcusplantarum, and Lactococcus raffinolactis. In some examples, theLactococcus lactis is Lactococcus lactis subsp. cremoris, Lactococcuslactis subsp. hordniae, or Lactococcus lactis subsp. lactis.

Exemplary Lactobacillus species include Lactobacillus acetotolerans,Lactobacillus acidophilus, Lactobacillus agilis, Lactobacillus algidus,Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillusamylophilus, Lactobacillus amylovorus, Lactobacillus animalis,Lactobacillus aviarius, Lactobacillus aviarius subsp. araffinosus,Lactobacillus aviarius subsp. aviarius, Lactobacillus bavaricus,Lactobacillus hifermenians, Lactobacillus brevis, Lactobacillusbuchneri, Lactobacillus bulgaricus, Lactobacillus carnis, Lactobacilluscasei, Lactobacillus casei subsp. alactosus, Lactobacillus casei subsp.casei, Lactobacillus casei subsp. pseudoplantarum, Lactobacillus caseisubsp. rhamnosus, Lactobacillus casei subsp. tolerans, Lactobacilluscatenaformis, Lactobacillus cellobiosus, Lactobacillus collinoides,Lactobacillus confusus, Lactobacillus coryniformis, Lactobacilluscoryniformis subsp. coryniformis, Lactobacillus coryniformis subsp.torquens, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacilluscurvatus subsp. curvatus, Lactobacillus curvatus subsp. melibiosus,Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus,Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckiisubsp. lactis, Lactobacillus divergens, Lactobacillus farciminis,Lactobacillus fermentum, Lactobacillus fornicalis, Lactobacillusfructivorans, Lactobacillus fructosus, Lactobacillus gallinarum,Lactobacillus gasseri, Lactobacillus graminis, Lactobacillushalotolerans, Lactobacillus hamsteri, Lactobacillus helveticus,Lactobacillus heterohiochii, Lactobacillus hilgardii, Lactobacillushomohiochii, Lactobacillus iners, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kandleri,Lactobacillus kefiri, Lactobacillus kefiranofaciens, Lactobacilluskefirgranum, Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillusleichmannii, Lactobacillus lindneri, Lactobacillus malefermentans,Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillusmanihotivorans, Lactobacillus minor, Lactobacillus minutus,Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii,Lactobacillus oris, Lactobacillus panis, Lactobacillus parabuchneri,Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei,Lactobacillus paracasei subsp. tolerans, Lactobacillus parakefiri,Lactobacillus paralimentarius, Lactobacillus paraplantarum,Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus piscicola,Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri,Lactobacillus rhamnosus, Lactobacillus rimae, Lactobacillus rogosae,Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus sakei subsp.camosus, Lactobacillus sakei subsp. sakei, Lactobacillus salivarius,Lactobacillus salivarius subsp. salicinius, Lactobacillus salivariussubsp. salivarius, Lactobacillus sanfranciscensis, Lactobacillussharpeae, Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillusuli, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus viridescens, Lactobacillus vitulinus, Lactobacillusxylosus, Lactobacillus yamanashiensis, Lactobacillus yamanashiensissubsp. mali, Lactobacillus yamanashiensis subsp. Yamanashiensis,Lactobacillus zeae, Bifidobacterium adolescentis, Bifidobacteriumangulatum, Bifidobacterium bifidum, Bifidobacterium breve,Bifidobacterium catenulatum, Bifidobacterium longum, and Bifidobacteriuminfantis. In some examples, the LAB is Lactococcus lactis (LL).

In further examples, the bacterium is selected from the group consistingof Enterococcus alcedinis, Enterococcus aquimarinus, Enterococcus asini,Enterococcus avium, Enterococcus caccae, Enterococcus camelliae,Enterococcus canintestini, Enterococcus canis, Enterococcuscasseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcusdevriesei, Enterococcus diestrammenae, Enterococcus dispar, Enterococcusdurans, Enterococcus eurekensis, Enterococcus faecalis, Enterococcusfaecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcushaemoperoxidus, Enterococcus hermanniensis, Enterococcus hirae,Enterococcus italicus, Enterococcus lactis, Enterococcus lemanii,Enterococcus malodoratus, Enterococcus moraviensis, Enterococcusmundtii, Enterococcus olivae, Enterococcus pallens, Enterococcusphoeniculicola, Enterococcus plantarum. Enterococcus pseudoavium,Enterococcus quebecensis, Enterococcus raffinosus, Enterococcus ralli,Enterococcus rivorum, Enterococcus rotai, Enterococcus saccharolvticus,Enterococcus silesiacus, Enterococcus solitarius, Enterococcussulfureus, Enterococcus termitis, Enterococcus thailandicus,Enterococcus ureasiticus, Enterococcus ureilyticus, Enterococcusviikkiensis, Enterococcus villorum, and Enterococcus xiangfangensis.

In further examples, the bacterium is selected from the group consistingof Streptococcus agalactiae, Streptococcus anginosus, Streptococcusbovis, Streptococcus canis, Streptococcus constellatus, Streptococcusdysgalactiae, Streptococcus equinus, Streptococcus iniae, Streptococcusintermedius, Streptococcus milleri, Streptococcus mitis, Streptococcusmutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcusperoris, Streptococcus pneumoniae, Streptococcus pseudopneumoniae,Streptococcus pyogenes. Streptococcus ratti, Streptococcus salivarius,Streptococcus tigurinus, Streptococcus thermophilus, Streptococcussanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcusuberis, Streptococcus vestibularis, Streptococcus viridans, andStreptococcus zooepidemicus.

In a particular aspect of the present invention, the Gram-positive foodgrade bacterial strain is Lactococcus lactis or any of its subspecies,including Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp.hordniae, and Lactococcus lactis subsp. lactis. In another aspect of thepresent invention, the recombinant Gram-positive bacterial strains is abiologically contained system, such as the plasmid free Lactococcuslactis strain MG1363, that lost the ability of normal growth and acidproduction in milk (Gasson, M. J. (1983) J. Bacteriol. 154:1-9); or thethreonine- and pyrimidine-auxotroph derivative L. lactis strains(Sorensen et al. (2000) Appl. Environ. Microbiol. 66:1253-1258; Glentinget al. (2002) 68:5051-5056).

In some examples according to any of the above embodiments, thebacterium is not E. coli.

In one embodiment of the present invention, the recombinant bacterialhost-vector system is a biologically contained system. Biologicalcontainment is known to the person skilled in the art and can berealized by the introduction of an auxotrophic mutation, e.g., asuicidal auxotrophic mutation such as the thyA mutation, or itsequivalents. Alternatively, the biological containment can be realizedat the level of the plasmid carrying the gene encoding a polypeptide,such as, for example, by using an unstable episomal construct, which islost after a few generations. Several levels of containment, such asplasmid instability and auxotrophy, can be combined to ensure a highlevel of containment, if desired.

Binding and Adherence Molecules

The terms “binding” and “adherence” are largely synonymous herein.Binding/adherence may be assessed through in vitro models with mammaliancells and/or biological surfaces such as mucus, fibronectin, collagen orother inanimate surfaces; and in vivo through, e.g., measurements ofcolonization, persistence, or as implied by biological effect.

Such binding/adherence is typically specific to a target molecule, cell,or site. Organisms may produce a wide variety of molecules (oftenpolypeptides, glycoproteins and carbohydrates) that facilitate bindingand adherence to other cells, inanimate objects, and cellular productssuch as mucus. Such molecules that facilitate binding and adherence havevarious degrees of specificity to a target molecule, typically anotherprotein, glycoprotein or carbohydrate. The expression of differentbinding molecules leads to preferential binding to different biologicalsurfaces. For example, the different cells of the GI tract expressdifferent surface molecules, and the frequency of specific surfacemolecules can differ along the GI tract. Thus, a bacterial cell canpreferentially bind to a specific host cell, or a specific region of theGI tract. Binding may be associated with increased colonization,delivery of target proteins at the target site, increased GI transittime and more. Conversely, binding to one site in the GI tract may beassociated with reduced binding to another GI site.

The natural variation of bacterial binding proteins can be supplementedby mutation, and by recombination to express motifs from other bindingmolecules, including from other organisms. Such recombinant bindingproteins may be expressed alone, or as a fusion proteins to providemultiple binding specificities in a single molecule.

For a protein to bind the bacterium to a biological surface, the bindingpolypeptide is typically exported from the cytoplasm and anchored to thesurface of the bacterium. In gram positive bacteria, such asLactococcus, adhesion molecules are typically expressed with (a) anN-terminal secretion signal to direct secretion through the cytoplasmicmembrane and (b) a C-terminal anchoring domain that anchors thepolypeptide to the cell wall (i.e., a “cell wall anchoring domain”).Without an anchoring domain, the polypeptide is released in theextracellular milieu.

For example, the trefoil factors (TFF) are secreted by animal cells,bind to mucus, and have a number of biological effects, includinghealing the mucus membrane. A bacterium can be engineered to secrete TFFinto the extracellular milieu to promote healing of the mucus membrane,by recombinantly adding a bacterial secretion signal to TFF. A bacteriumcan also be engineered to have mucus binding properties, by adding botha secretion and anchor signal to TFF.

Cell-Adherence Polypeptide

In some embodiments of the present disclosure, the microorganism (e.g.,bacterium) contains an exogenous nucleic acid encoding a fusion proteincontaining a cell-adherence polypeptide. Any polypeptide exhibitingcell-adherence properties, e.g., binding to intestinal cells orcell-lines thereof (e.g., Caco-2, IEC-18, or HT29-MTX cells) are usefulin the context of the present disclosure. Cell-adherence capabilitiesmay be measured using art-recognized methods, such as those disclosedherein. In some examples, the cell-adherence polypeptide is selectedfrom cell and mucus-binding protein A (CmbA) (see, e.g., Jensen et al.,Microbiology 2014, 160(4):671-681), mucus binding protein or mub domainproteins (Mub) (see, e.g., Boekhorst et al., Microbiology 2006,152(1):273-280), mucus adhesion promoting protein (MapA) (see, e.g.,Miyoshi et al., Biosci. Biotechnol. Biochem. 2006, 70(7):1622-8),lactococcal mucin binding protein (MpbL) (see, e.g., Lukić et al., Appl.Environ. Microbiol. 2012, 78(22):7993-8000). In some examples, thefusion protein may include a cell-wall anchor peptide, such asStaphylococcus aureus protein A anchor fragment (SpaX) (see, e.g.,Steidler et al., Appl. Environ. Microbiol. 1998, 64(1):342-5). All ofthe above disclosures are incorporated herein by reference in theirentirety. In some examples, the cell-adherence polypeptide is a CmbApolypeptide, such as CmbA from Lactobacillus reuteri. See, e.g., ATCCPTA6474, e.g., as disclosed in Jensen et al., supra.

In some examples according to any of the above embodiments, thecell-adherence polypeptide is a CmbA polypeptide having an amino acidsequence that is at least 90%, at least 92%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.In other examples according to any of the above embodiments, thecell-adherence polypeptide is a CmbA polypeptide encoded by an exogenousnucleic acid sequence that is at least 90%, at least 92%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% identical toSEQ ID NO: 2.

Mucin-Binding Polypeptide

In some embodiments of the present disclosure the microorganism (e.g.,bacterium) contains an exogenous nucleic acid encoding a fusion proteincontaining a mucin-binding polypeptide. Any polypeptide exhibitingmucin-binding properties, e.g., binding to mucin preparations in vitro,are useful in the context of the present disclosure. Mucin-bindingcapabilities may be measured using art-recognized methods, such as thosedisclosed herein. Exemplary mucin-binding polypeptides include trefoilfactor (TFF) polypeptides (e.g., TFF1, TFF2, or TFF3) (see, e.g.,Caluwaerts, S. et al., Oral. Oncol. 2010, 46:564-570) and MucBPpolypeptides (see, e.g., Lukic et al, Appl. Environ. Microbiol. 2012,78(22):7993-8000). In some examples, the current disclosure provides amicroorganism (e.g., a bacterium) comprising an exogenous nucleic acidencoding a fusion protein, wherein the fusion protein contains acell-adherence polypeptide (e.g., a CmbA polypeptide) and amucin-binding polypeptide (e.g., a TFF polypeptide). In some examples,the TFF polypeptide is a human TFF polypeptide (e.g., hTFF1, hTFF2, orhTFF3). In other examples according to any of the above embodiments, themucin-binding polypeptide is a human TFF3 polypeptide having an aminoacid sequence that is at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3. In other examples according to any of the above embodiments, themucin-binding polypeptide is a human TFF3 polypeptide encoded by anexogenous nucleic acid sequence that is at least 90%, at least 92%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 4. In some examples, the mucin-bindingpolypeptide is a fish TFF polypeptide, or an amphibian TFF polypeptide.In yet other examples, the mucin-binding polypeptide includes (orconsists of) a trefoil-like domain, such as those disclosed in Fujita etal., Mol. Reprod. Dev. 2006, 75(7):1217-1228.

Constructs

In some embodiments the microorganism (e.g., bacterium, such asLactococcus lactis) comprises an expression vector capable of expressingthe fusion protein and optionally a therapeutic polypeptide. Forexample, the fusion protein is exposed on the cell surface underconditions present at the mucosa, e.g., in the gastrointestinal tract.The microorganism (e.g., bacterium) can comprise expression vectorscapable of expressing the fusion protein, such that the fusion proteinis exposed on the cell surface to a degree sufficient to provide thedesired GI retention. One of skill in the art may adjust the amount ofmicroorganisms (e.g., bacterium) provided to the subject to deliver thedesired amount of therapeutic polypeptide.

Usually, the expression system will comprise a genetic constructcomprising at least one nucleotide sequence encoding at least one fusionprotein, e.g., operably linked to a promoter capable of directingexpression of the sequence(s) in the hosting microorganism. Suitably thefusion protein to be expressed can be encoded by a nucleic acid sequencethat is adapted to the preferred codon usage of the host. The constructmay further contain (all) other suitable element(s), includingenhancers, transcription initiation sequences, signal sequences,reporter genes, transcription termination sequences, etc., operable inthe selected host, as is known to the person skilled in the art.

In some examples, the construct is in a form suitable for transformationof the host and/or in a form that can be stably maintained in the host,such as a vector, plasmid or mini-chromosome. Suitable vectorscomprising nucleic acid for introduction into microorganisms (e.g.,bacteria) can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, enhancer sequences, marker genes and other sequences asappropriate. Vectors may be plasmids, viral (e.g., phage or phagemid),as appropriate. For further details see, for example, Molecular Cloning:a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold SpringHarbor Laboratory Press, the relevant disclosure of which isincorporated herein by reference.

Many known techniques and protocols for the manipulation of nucleicacids, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail in ShortProtocols in Molecular Biology, Second Edition, Ausubel et al., eds.,John Wiley & Sons, 1992, the relevant disclosure of which isincorporated herein by reference. In one embodiment, the coding sequencefor the fusion protein is contained in an operon, i.e., a nucleic acidconstruct for poly-cistronic expression. In an operon, transcriptionfrom the promoter results in a mRNA which comprises more than one codingsequence, each with its own suitably positioned ribosome binding siteupstream. Thus, more than one polypeptide can be translated from asingle mRNA. Use of an operon enables expression of the fusion proteinand a therapeutic polypeptide to be coordinated. Polycistronicexpression systems in bacterial host cells are described, e.g., in U.S.Patent Application No. 2014/0105863 to Vanden-Broucke et al., which isincorporated herein by reference in its entirety.

In an embodiment the present invention relates to stably transfectedmicroorganisms (e.g., bacteria). In some examples, the presentdisclosure provides microorganisms (e.g., bacteria), in which theexogenous nucleic acid encoding the fusion protein has been integratedinto the host cell's chromosome. Techniques for establishing stablytransfected microorganisms are known in the art. For instance, thenucleic acid encoding the fusion protein may be cloned into the host'schromosome via homologous recombination. In some examples, an essentialgene of the host is disrupted by the homologous recombination event,such as deletion of the gene, one or more amino acid substitutionsleading to an inactive form of the protein encoded by the essentialgene, or to a frameshift mutation resulting in a truncated form of theprotein encoded by the essential gene. In an embodiment, the essentialgene is the thyA gene. An exemplary technique is described, e.g., in WO02/090551, which is incorporated herein by reference in its entirety.The transforming plasmid can be any plasmid, as long as it cannotcomplement the disrupted essential gene, e.g., thyA gene. The plasmidmay be self-replicating, may carry one or more genes of interest, andmay carry one or more resistance markers. In some examples, the plasmidis an integrative plasmid (i.e., integration plasmid). Such integrativeplasmid may be used to disrupt the essential gene, by causingintegration at the locus of the essential gene, e.g., thyA site, becauseof which the function of the essential gene, e.g., the thyA gene, isdisrupted. In some examples, the essential gene, such as the thyA gene,is replaced by double homologous recombination by a cassette comprisingthe gene or genes of interest, flanked by targeting sequences thattarget the insertion to the essential gene, such as the thyA targetsite. It will be appreciated that that these targeting sequences aresufficiently long and sufficiently homologous to enable integration ofthe gene of interest into the target site.

The genetic construct encoding the fusion protein may be present in thehost cell extra-chromosomally, e.g., autonomously replicating using anown origin of replication, or may be integrated into the microbialgenomic DNA, e.g., bacterial or yeast chromosome, e.g., Lactococcuschromosome. In the latter case, a single copy or multiple copies of thenucleic acid may be integrated; the integration may occur at a randomsite of the chromosome or, as described above, at a predetermined sitethereof, such as in the thyA locus of Lactococcus, e.g., Lactococcuslactis.

Hence, in some embodiments, the genetic construct encoding the fusionprotein may further comprises sequences configured to effect insertionof the genetic construct into the chromosome of a host cell. In someexamples, insertion of the genetic construct into particular siteswithin a microbial genome, e.g., chromosome of a host cell may befacilitated by homologous recombination. For instance, a geneticconstruct of the present disclosure may comprise one or more regions ofhomology to the site of integration within the chromosome, of the hostcell. The sequence at the chromosome site may be natural, i.e., asoccurring in nature, or may be an exogenous sequence introduced byprevious genetic engineering.

In some examples, the region(s) of homology may be at least 50 basepairs (bp), 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp 700 bp, 800bp, 900 bp, 1000 bp, or more.

In one example, two regions of homology may be included, one flankingeach side of the relevant expression units present in the geneticconstruct. Such configuration may advantageously insert the relevantsequences into host cells. Methods for homologous recombination,especially in bacterial hosts, and selecting for recombinants, aregenerally known in the art.

Transformation methods of microorganisms are known to the person skilledin the art, such as for instance protoplast transformation andelectroporation.

High degrees of expression can be achieved by using homologousexpression and/or secretion signals on the expression vectors present inthe microorganism, e.g., Lactococcus lactis. Expression signals will beapparent to the person skilled in the art. The expression vector can beoptimized for expression depending on the microorganism it isincorporated into. For instance, specific expression vectors that gavesufficient levels of expression in Lactococcus, Lactobacillus lactis,casei and plantarum are known. Moreover, systems are known which havebeen developed for the expression of heterologous antigens in thenon-pathogenic, non-colonizing, non-invasive food-grade bacteriumLactococcus lactis (see UK patent GB2278358B, which is incorporatedherein by reference). In some examples, construct of the presentdisclosure comprises the multi-copy expression vector described inPCT/NL95/00135 (WO-A-96/32487), in which the nucleotide sequenceencoding the fusion protein has been incorporated. Such a construct isparticularly suitable for expression of a desired polypeptide in alactic acid bacterium, in particular in a Lactobacillus, at a high levelof expression, and also can be used to direct the expressed product tothe surface of the bacterial cell. The constructs (e.g., ofPCT/NL95/00135) may be characterized in that the nucleic acid sequenceencoding the fusion protein is preceded by a 5′ non-translated nucleicacid sequence comprising at least the minimal sequence required forribosome recognition and RNA stabilization. This can be followed by atranslation initiation codon which may be (immediately) followed by afragment of at least 5 codons of the 5′ terminal part of the translatednucleic acid sequence of a gene of a lactic acid bacterium or astructural or functional equivalent of the fragment. The fragment mayalso be controlled by the promoter. The contents of PCT/NL95/00135,including the differing embodiments disclosed therein, and all otherdocuments mentioned in this specification, are incorporated herein byreference. One aspect of the present invention provides a method whichpermits the high level regulated expression of heterologous genes in thehost and the coupling of expression to secretion. In another embodiment,the T7 bacteriophage RNA polymerase and its cognate promoter are used todevelop a powerful expression system according to WO 93/17117, which isincorporated herein by reference. In one embodiment, the expressionplasmid is derived from pT1 NX.

In some embodiments, a promoter employed in accordance with the presentdisclosure is expressed constitutively in the bacterium. The use of aconstitutive promoter avoids the need to supply an inducer or otherregulatory signal for expression to take place. For example, thepromoter directs expression at a level at which the bacterial host cellremains viable, i.e., retains some metabolic activity, even if growth isnot maintained. Advantageously, such expression may be at a low level.For example, where the expression product accumulates intracellularly,the level of expression may lead to accumulation of the expressionproduct at less than about 10% of cellular protein, or about or lessthan about 5%, for example about 1-3%. The promoter may be homologous tothe bacterium employed, i.e., one found in that bacterium in nature. Forexample, a Lactococcal promoter may be used in a Lactococcus. Anexemplary promoter for use in Lactococcus lactis (or other Lactococci)is “P1” derived from the chromosome of Lactococcus lactis (Waterfield, NR, Lepage, R W F, Wilson, P W, et al. (1995). “The isolation oflactococcal promoters and their use in investigating bacterialluciferase synthesis in Lactococcus lactis” Gene 165(1):9-15). Anotherexample of a promoter is the usp4 promoter. Other useful promoters aredescribed in U.S. Pat. No. 8,759,088 to Steidler et al., and in U.S.Patent Application No. 2014/0105863 to Vandenbroucke et al., thedisclosures of which are incorporated herein by reference in theirentirety.

The nucleic acid construct or constructs may comprise a secretory signalsequence. Thus, in some embodiments the nucleic acid encoding the fusionprotein may provide for secretion of the polypeptides, e.g., byappropriately coupling a nucleic acid sequence encoding a signalsequence to the nucleic acid sequence encoding the polypeptide). Abilityof a bacterium harboring the nucleic acid to secrete the antigen may betested in vitro in culture conditions which maintain viability of theorganism. Exemplary secretory signal sequences include any of those withactivity in Gram-positive organisms such as Bacillus, Clostridium andLactobacillus. Such sequences may include the α-amylase secretion leaderof Bacillus amyloliquefaciens or the secretion leader of theStaphylokinase enzyme secreted by some strains of Staphylococcus, whichis known to function in both Gram-positive and Gram-negative hosts (see“Gene Expression Using Bacillus,” Rapoport (1990) Current Opinion inBiotechnology 1:21-27), or leader sequences from numerous other Bacillusenzymes or S-layer proteins (see pp 341-344 of Harwood and Cutting,“Molecular Biological Methods for Bacillus,” John Wiley & Co. 1990). Inone embodiment, said secretion signal is derived from usp45 (VanAsseldonk et al., Mol. Gen. Genet. 1993, 240:428-434). In someembodiments, the fusion protein is constitutively secreted.

Formulations and Regimens

In the methods of the present disclosure, multiple therapeuticpolypeptides may be expressed by the same or different microorganisms.For example, (a) PAL and amino acid decarboxylase; (b) a T1D specificantigen such as PINS and a Treg activating cytokine such as IL-2 orIL-10 (c) a gluten antigen and IL-2 or IL-10, and the like. If expressedin separate organisms, one or preferably both bacteria will expressmucin and/or cell-binding factors. When the two polypeptides areexpressed by different microorganisms, those may be administered to thesubject in the same (e.g., combined) formulation or may be administeredin separate (e.g., different) formulations. Separate formulations may beadministered at the same time or at different time points. For example,the use of first and second therapeutic polypeptide producingmicroorganisms in their respective formulations can be administered tothe subject simultaneously or may be administered sequentially, e.g.,with a rest period between administrations.

In some embodiments, the first and second therapeutic polypeptideproducing microorganisms are administered simultaneously. In someexamples, according to these embodiments, the first therapeuticpolypeptide microorganism, and the second therapeutic polypeptidemicroorganism are comprised in the same pharmaceutical formulation, orin more than one pharmaceutical formulation taken at the same time. Insome embodiments, the two bioactive polypeptides are delivered to thesubject using a microorganism producing both the IL-2 and theT1D-specific antigen.

In some embodiments, the microorganism will be administered, once,twice, three, four, five, or six times daily, e.g., using an oralformulation. In some embodiments, the microorganisms are administeredevery day, every other day, once per week, twice per week, three timesper week, or four times per week. In other embodiments, treatment occursonce every two weeks. In other embodiments, treatment occurs once everythree weeks. In other embodiments, treatment occurs once per month.

The duration of a treatment cycle for the method is, for example, 7 daysto the subject's lifetime, as needed to treat or reverse disease, orprevent relapse. In some embodiments, a treatment cycle lasts for about30 days to about 2 years. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 1.5 years. In otherembodiments, the subject will have a treatment cycle that lasts from 30days to 1 year. In other embodiments, the subject will have a treatmentcycle that lasts from 30 days to 11 months. In other embodiments, thesubject will have a treatment cycle that lasts from 30 days to 10months. In other embodiments, the subject will have a treatment cyclethat lasts from 30 days to 9 months. In other embodiments, the subjectwill have a treatment cycle that lasts from 30 days to 8 months. Inother embodiments, the subject will have a treatment cycle that lastsfrom 30 days to 7 months. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 6 months. In otherembodiments, the subject will have a treatment cycle that lasts from 30days to 5 months. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 4 months. In otherembodiments, the subject will have a treatment cycle that lasts from 30days to 3 months. In other embodiments, the subject will have atreatment cycle that lasts from 30 days to 2 months.

Daily maintenance doses can be given for a period clinically desirablein the subject, for example from 1 day up to several years (e.g. for thesubject's entire remaining life); for example from about (2, 3 or 5days, 1 or 2 weeks, or 1 month) upwards and/or for example up to about(5 years, 1 year, 6 months, 1 month, 1 week, or 3 or 5 days).Administration of the daily maintenance dose for about 3 to about 5 daysor for about 1 week to about 1 year is typical. Unit doses can beadministered from twice daily to once every two weeks until atherapeutic effect is observed.

The microorganisms producing the first and second therapeuticpolypeptide may be delivered in mono- or combination therapy for thetreatment of the disease. In some embodiments, the compositions of thepresent disclosure include additional therapeutically active agents. Insome embodiments, the compositions of the present disclosure, andtreatment of the subject, does not involve other active components,e.g., does not involve additional immune-modulating substances, such asantibodies (e.g., anti-CD3 for treatment of T1D). Thus, in someexamples, the pharmaceutical compositions of the present disclosureconsist essentially of the microorganism as described herein (expressingthe therapeutic IL-2 and antigen polypeptides), and a pharmaceuticallyacceptable carrier.

Pharmaceutical Compositions and Carriers

Microorganisms (e.g., bacteria or yeast as described herein) may beadministered in pure form, combined with other active ingredients,and/or combined with pharmaceutically acceptable (i.e., nontoxic)excipients or carriers. The term “pharmaceutically acceptable” is usedherein in accordance with its art-recognized meaning and refers tocarriers that are compatible with the other ingredients of apharmaceutical composition, and are not deleterious to the recipientthereof.

The compositions of the present invention can be prepared in any knownor otherwise effective dosage or product form suitable for use inproviding systemic delivery of the microorganism (e.g., bacteria) to themucosa, which would include pharmaceutical compositions and dosage formsas well as nutritional product forms.

In some embodiments, the formulation is an oral formulation orpharmaceutical composition. In some examples according to thisembodiment, the formulation or pharmaceutical composition comprises thenon-pathogenic microorganism in a dry-powder form (e.g., freeze-driedform) or in compacted form thereof, optionally in combination with otherdry carriers. Oral formulations will generally include an inert diluentcarrier or an edible carrier.

In some examples, the oral formulation comprises a coating or utilizesan encapsulation strategy, which facilitates the delivery of theformulation into the intestinal tract, and/or allows the microorganismbe released and hydrated in the intestinal tract (e.g., the ileum, smallintestine, or the colon). Once the microorganism is released from theformulation and sufficiently hydrated, it begins expressing thebioactive polypeptide, which is subsequently released into thesurroundings, or expressed on the surface of the microorganism. Suchcoating and encapsulation strategies (i.e., delayed-release strategies)are known to those of skill in the art. See, e.g., U.S. Pat. No.5,972,685; WO 2000/18377; and WO 2000/22909, the disclosures of whichare incorporated herein by reference in their entirety.

In some embodiments, the disclosure provides a pharmaceuticalcomposition comprising the microorganism (e.g., the non-pathogenicbacteria) in a lyophilized or freeze dried form, optionally inconjunction with other components, such as dextrans, sodium glutamate,and polyols. Exemplary freeze dried compositions are described, e.g., inU.S. Patent Application No. 2012/0039853 to Corveleyn et al., thedisclosure of which is incorporated herein by reference in its entirety.Exemplary formulations comprise freeze-dried bacteria (e.g., atherapeutically effective amount of the bacteria) and a pharmaceuticallyacceptable carrier. Freeze-dried bacteria may be prepared in the form ofcapsules, tablets, granulates and powders, each of which may beadministered orally. Alternatively, freeze-dried bacteria may beprepared as aqueous or oily suspensions in suitable media, orlyophilized bacteria may be suspended in a suitable medium, such as adrink, just prior to use.

For oral administration, the formulation may be a gastro-resistant oraldosage form. For example, the oral dosage form (e.g., capsules, tablets,pellets, micro-pellets, granulates, and the like) may be coated with athin layer of excipient (usually polymers, cellulosic derivatives and/orlipophilic materials) that resists dissolution or disruption in thestomach, but not in the intestine, thereby allowing transit through thestomach in favor of disintegration, dissolution and absorption in theintestine (e.g., the small intestine, or the colon). In some examples,oral formulations may include compounds providing controlled release,sustained release, or prolonged release of the microorganism, andthereby provide controlled release of the desired protein encodedtherein. These dosage forms (e.g., tablets or capsules) typicallycontain conventional and well known excipients, such as lipophilic,polymeric, cellulosic, insoluble, swellable excipients. Controlledrelease formulations may also be used for any other delivery sitesincluding intestinal, colon, bioadhesion or sublingual delivery (i.e.,dental mucosal delivery) and bronchial delivery. When the compositionsof the invention are to be administered rectally or vaginally,pharmaceutical formulations may include suppositories and creams. Inthis instance, the host cells are suspended in a mixture of commonexcipients also including lipids. Each of the aforementionedformulations are well known in the art and are described, for example,in the following references: Hansel et al. (1990, Pharmaceutical dosageforms and drug delivery systems, 5th edition, William and Wilkins);Chien 1992, (Novel drug delivery system, 2nd edition, M. Dekker);Prescott et al. (1989, Novel drug delivery, J. Wiley & Sons); Gazzanigaet al., (1994, Oral delayed release system for colonic specificdelivery, Int. J. Pharm. 108:77-83).

In some embodiments, the oral formulation includes compounds that canenhance mucosal delivery and/or mucosal uptake of the bioactivepolypeptides expressed by the microorganism. In other examples, theformulation includes compounds, which enhance the viability of themicroorganism within the formulation, and/or once released.

The bacteria of the invention can be suspended in a pharmaceuticalformulation for administration to the human or animal having the diseaseto be treated. Such pharmaceutical formulations include but are notlimited to live Gram-positive bacteria and a medium suitable foradministration. The bacteria may be lyophilized in the presence ofcommon excipients such as lactose, other sugars, alkaline and/or alkaliearth stearate, carbonate and/or sulphate (e.g., magnesium stearate,sodium carbonate and sodium sulphate), kaolin, silica, flavorants andaromas. Bacteria so-lyophilized may be prepared in the form of capsules,tablets, granulates and powders (e.g., a mouth rinse powder), each ofwhich may be administered by the oral route. Alternatively, someGram-positive bacteria may be prepared as aqueous suspensions insuitable media, or lyophilized bacteria may be suspended in a suitablemedium just prior to use, such medium including the excipients referredto herein and other excipients such as glucose, glycine and sodiumsaccharinate.

In some examples, the microorganism is locally delivered to thegastrointestinal tract of the subject using any suitable method. Forexample, microsphere delivery systems could be employed to enhancedelivery to the gut. Microsphere delivery systems include microparticleshaving a coating that provides localized release into thegastrointestinal tract of the subject (e.g., controlled releaseformulations such as enteric-coated formulations and colonicformulations).

For oral administration, gastroresistant oral dosage forms may beformulated, which dosage forms may also include compounds providingcontrolled release of the Gram-positive bacteria and thereby providecontrolled release of the desired protein encoded therein (e.g., IL-2).For example, the oral dosage form (including capsules, tablets, pellets,granulates, powders) may be coated with a thin layer of excipient (e.g.,polymers, cellulosic derivatives and/or lipophilic materials) thatresists dissolution or disruption in the stomach, but not in theintestine, thereby allowing transit through the stomach in favor ofdisintegration, dissolution and absorption in the intestine.

The oral dosage form may be designed to allow slow release of theGram-positive bacteria and of the produced exogenous proteins, forinstance as controlled release, sustained release, prolonged release,sustained action tablets or capsules. These dosage forms usually containconventional and well-known excipients, such as lipophilic, polymeric,cellulosic, insoluble, swellable excipients. Such formulations arewell-known in the art and are described, for example, in the followingreferences: Hansel et al., Pharmaceutical dosage forms and drug deliverysystems, 5th edition, William and Wilkins, 1990; Chien 1992, Novel drugdelivery system, 2nd edition, M. Dekker; Prescott et al., Novel drugdelivery, J. Wiley & Sons, 1989; and Gazzaniga et al., Int. J. Pharm.108:77-83 (1994).

The pharmaceutical dosage form (e.g. capsule) is coated withpH-dependent Eudragit polymers to obtain gastric juice resistance andfor the intended delivery at the terminal ileum and colon, where thepolymers dissolve at pH 6.5. By using other Eudragit polymers or adifferent ratio between the polymers, the delayed release profile couldbe adjusted, to release the bacteria for example in the duodenum orjejunum.

Pharmaceutical compositions contain at least one pharmaceuticallyacceptable carrier. Non-limiting examples of suitable excipients,diluents, and carriers include preservatives, inorganic salts, acids,bases, buffers, nutrients, vitamins, fillers and extenders such asstarch, sugars, mannitol, and silicic derivatives; binding agents suchas carboxymethyl cellulose and other cellulose derivatives, alginates,gelatin, and polyvinyl pyrolidone; moisturizing agents such asglycerol/disintegrating agents such as calcium carbonate and sodiumbicarbonate; agents for retarding dissolution such as paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents such as acetyl alcohol, glycerol monostearate; adsorptivecarriers such as kaolin and bentonite; carriers such as propylene glycoland ethyl alcohol, and lubricants such as talc, calcium and magnesiumstearate, and solid polyethyl glycols.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. Tablets, pills, capsules,troches and the like can contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a dispersing agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate; a glidant such ascolloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carriersuch as a fatty oil. In addition, dosage unit forms can contain variousother materials that modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or enteric agents. Further, a syrupmay contain, in addition to the active compounds, sucrose as asweetening agent and certain preservatives, dyes, colorings, andflavorings. It will be appreciated that the form and character of thepharmaceutically acceptable carrier is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

Alternative preparations for administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are dimethylsulfoxide, alcohols, propylene glycol,polyethylene glycol, vegetable oils such as olive oil and injectableorganic esters such as ethyl oleate. Aqueous carriers include mixturesof alcohols and water, buffered media, and saline. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, anti-oxidants, chelating agents, inert gases, and thelike. Various liquid formulations are possible for these deliverymethods, including saline, alcohol, DMSO, and water based solutions.

Oral aqueous formulations include excipients, such as pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate and/or the like. Thesecompositions take the form of solutions such as mouthwashes and mouthrinses, further comprising an aqueous carrier such as for example water,alcoholic/aqueous solutions, saline solutions, parenteral vehicles suchas sodium chloride, Ringer's dextrose, and the like.

Aqueous mouthwash formulations are well-known to those skilled in theart. Formulations pertaining to mouthwashes and oral rinses arediscussed in detail, for example, in U.S. Pat. Nos. 6,387,352,6,348,187, 6,171,611, 6,165,494, 6,117,417, 5,993,785, 5,695,746,5,470,561, 4,919,918, U.S. Patent Appl. No. 2004/0076590, U.S. PatentAppl. No. 2003/0152530, and U.S. Patent Appl. No. 2002/0044910, each ofwhich is herein specifically incorporated by reference into this sectionof the specification and all other sections of the specification.

Other additives may be present in the formulations of the presentdisclosure, such as flavoring, sweetening or coloring agents, orpreservatives. Mint, such as from peppermint or spearmint, cinnamon,eucalyptus, citrus, cassia, anise and menthol are examples of suitableflavoring agents. Flavoring agents can be present in the oralcompositions in an amount in the range of from 0 to 3%, e.g., up to 2%,such as up to 0.5%, e.g., around 0.2%, in the case of liquidcompositions.

Sweeteners include artificial or natural sweetening agents, such assodium saccharin, sucrose, glucose, saccharin, dextrose, levulose,lactose, mannitol, sorbitol, fructose, maltose, xylitol, thaumatin,aspartame, D-tryptophan, dihydrochalcones, acesulfame, and anycombinations thereof, which may be present in an amount in the range offrom about 0 to 2%, e.g., up to 1% w/w, such as 0.05 to 0.3% w/w of theoral composition.

Coloring agents are suitable natural or synthetic colors, such astitanium dioxide or CI 42090, or mixtures thereof. Coloring agents canbe present in the compositions in an amount in the range of from 0 to3%; e.g., up to 0.1%, such as up to 0.05%, e.g., about 0.005-0.0005%, inthe case of liquid compositions. In some examples, sodium benzoate isadded as a preservative, e.g., in concentrations insufficientsubstantially to alter the pH of the composition, otherwise the amountof buffering agent may need to be adjusted to arrive at the desired pH.

Other optional ingredients include humectants, surfactants (non-ionic,cationic or amphoteric), thickeners, gums and binding agents. Ahumectant adds body to the formulation and retains moisture in adentifrice composition. In addition, a humectant helps to preventmicrobial deterioration during storage of the formulation. It alsoassists in maintaining phase stability and provides a way to formulate atransparent or translucent dentifrice.

Suitable humectants include glycerin, xylitol, glycerol and glycols suchas propylene glycol, which may be present in an amount of up to 50% w/weach, but total humectant is e.g., not more than about 60-80% w/w of thecomposition. For example, liquid compositions may comprise up to about30% glycerin plus up to about 5%, or about 2% w/w xylitol. In someexamples, surfactants are not anionic and may include polysorbate 20 orcocoamidobetaine or the like in an amount up to about 6%, or about 1.5to 3%, w/w of the composition.

In some examples, when the oral compositions of the invention is in aliquid form, it a film-forming agent that may be added up to about 3%w/w of the oral composition, such as in the range of from 0 to 0.1%, orabout 0.001 to 0.01%, such as about 0.005% w/w of the oral composition.Suitable film-formers include (in addition to sodium hyaluronate) thosesold under the tradename Gantrez.

Liquid nutritional formulations for oral or enteral administration maycomprise one or more nutrients such as fats, carbohydrates, proteins,vitamins, and minerals. Many different sources and types ofcarbohydrates, lipids, proteins, minerals and vitamins are known and canbe used in the nutritional liquid embodiments of the present invention,provided that such nutrients are compatible with the added ingredientsin the selected formulation, are safe and effective for their intendeduse, and do not otherwise unduly impair product performance.

These nutritional liquids can be formulated with sufficient viscosity,flow, or other physical or chemical characteristics to provide a moreeffective and soothing coating of the mucosa while drinking oradministering the nutritional liquid. These nutritional embodiments mayalso represent a balanced nutritional source suitable for meeting thesole, primary, or supplemental nutrition needs of the individual.

Non-limiting examples of suitable nutritional liquids are described inU.S. Pat. No. 5,700,782 (Hwang et al.); U.S. Pat. No. 5,869,118 (Morriset al.); and U.S. Pat. No. 5,223,285 (DeMichele et al.), whichdescriptions are incorporated herein by reference.

Nutritional proteins suitable for use herein can be hydrolyzed,partially hydrolyzed or non-hydrolyzed, and can be derived from anyknown or otherwise suitable source such as milk (e.g., casein, whey),animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable (e.g.,soy), or combinations thereof.

Fats or lipids suitable for use in the nutritional liquids include, butare not limited to, coconut oil, soy oil, corn oil, olive oil, saffloweroil, high oleic safflower oil, MCT oil (medium chain triglycerides),sunflower oil, high oleic sunflower oil, structured triglycerides, palmand palm kernel oils, palm olein, canola oil, marine oils, cottonseedoils, and combinations thereof. Carbohydrates suitable for use in thenutritional liquids may be simple or complex, lactose-containing orlactose-free, or combinations thereof. Non-limiting examples of suitablecarbohydrates include hydrolyzed corn starch, maltodextrin, glucosepolymers, sucrose, corn syrup, corn syrup solids, rice-derivedcarbohydrate, glucose, fructose, lactose, high fructose corn syrup andindigestible oligosaccharides such as fructo-oligosaccharides (FOS), andcombinations thereof.

The nutritional liquids may further comprise any of a variety ofvitamins, non-limiting examples of which include vitamin A, vitamin D,vitamin E, vitamin K, thiamine, riboflavin, pyridoxine, vitamin B12,niacin, folic acid, pantothenic acid, biotin, vitamin C, choline,inositol, salts and derivatives thereof, and combinations thereof.

The nutritional liquids may further comprise any of a variety ofminerals known or otherwise suitable for us in patients at risk of orsuffering from T1D, non-limiting examples of which include calcium,phosphorus, magnesium iron, selenium, manganese, copper, iodine, sodium,potassium, chloride, and combinations thereof.

The microorganisms and in particular the yeast and bacteria of thepresent invention can also be formulated as elixirs or solutions forconvenient oral or rectal administration or as solutions appropriate forparenteral administration, for instance by intramuscular, subcutaneousor intravenous routes. Additionally, the nucleoside derivatives are alsowell suited for formulation as a sustained or prolonged release dosageforms, including dosage forms that release active ingredient only or ina particular part of the intestinal tract, e.g., over an extended orprolonged period of time to further enhance effectiveness. The coatings,envelopes, and protective matrices in such dosage forms may be made, forexample, from polymeric substances or waxes well known in thepharmaceutical arts.

The compositions of the present invention include pharmaceutical dosageforms such as lozenges, troches or pastilles. These are typicallydiscoid-shaped solids containing the active ingredient in a suitablyflavored base. The base may be a hard sugar candy, glycerinated gelatin,or the combination of sugar with sufficient mucilage to give it form.Troches are placed in the mouth where they slowly dissolve, liberatingthe active ingredient for direct contact with the mucosa.

The troche embodiments of the present invention can be prepared, forexample, by adding water slowly to a mixture of the powdered active,powdered sugar, and a gum until a pliable mass is formed. A 7% acaciapowder can be used to provide sufficient adhesiveness to the mass. Themass is rolled out and the troche pieces cut from the flattened mass, orthe mass can be rolled into a cylinder and divided. Each cut or dividedpiece is shaped and allowed to dry, to thus form the troche dosage form.

If the active ingredient is heat labile, it may be made into a lozengepreparation by compression. For example, the granulation step in thepreparation is performed in a manner similar to that used for anycompressed tablet. The lozenge is made using heavy compression equipmentto give a tablet that is harder than usual as it is desirable for thedosage form to dissolve or disintegrate slowly in the mouth. In someexamples, ingredients are selected to promote slow-dissolvingcharacteristics.

In a particular formulation of the present invention, the microorganismswill be incorporated in a bioadhesive carrier containing pregelatinizedstarch and cross-linked poly (acrylic acid) to form a bioadhesive tabletand a bioadhesive gel suitable for buccal application (i.e., havingprolonged bioadhesion and sustained drug delivery). For example, apowder mixture of non-pathogenic and non-invasive bacterium according tothe invention), bioadhesive polymers (pregelatinized starch andcross-linked poly (acrylic acid) coprocessed via spray drying), sodiumstearyl fumarate (lubricant) and silicon dioxide (glidant) is processedinto tablets (weight: 100 mg; diameter: 7 mm). The methods for theproduction of these tablets are well known to the person skilled in theart and has been described before for the successful development ofbioadhesive tablets containing various drugs (miconazol, testosterone,fluoride, ciprofloxacin) (Bruschi M. L. and de Freitas O., DrugDevelopment and Industrial Pharmacy, 2005 31:293-310). All excipientmaterials are commercially available in pharmaceutical grades.

To optimize the formulation, the drug load in the tablets and the ratiobetween starch and poly (acrylic acid) will be varied. Based on previousresearch, the maximum drug load in the coprocessed bioadhesive carrieris about 60% (w/w) and the starch/poly (acrylic acid) ratio can bevaried between 75/25 and 95/5 (w/w). During the optimization study thebioadhesive properties of the tablets and the drug release from thetablets are the main evaluation parameters, with the standard tabletproperties (hardness, friability) as secondary evaluation criteria.

The bacteria are incorporated into an aqueous dispersion ofpregelatinized starch and cross-linked poly (acrylic acid). This polymerdispersion is prepared via a standard procedure using a high shearmixer.

Similar to the tablet, the drug load of the gel and the starch/poly(acrylic acid) ratio need to be optimized in order to obtain a gelhaving optimal adherence to the esophageal mucosa. For a gel, theconcentration of the polymers in the dispersion is an additionalvariable as it determines the viscosity of the gel, hence itsmuco-adhesive properties.

A model to screen the bioadhesive properties of polymer dispersions tothe mucosa of esophagus has been described in detail by Batchelor et al.(Int. J. Pharm., 238:123-132, 2002).

Other routes and forms of administration include food preparationscontaining the live microorganisms. In some examples, the bioactivepolypeptide-expressing microorganism can be included into a dairyproduct.

The pharmaceutical compositions of the present invention can be preparedby any known or otherwise effective method for formulating ormanufacturing the selected dosage form. For example, the microorganismscan be formulated along with common, e.g., pharmaceutically acceptablecarriers, such as excipients and diluents, formed into oral tablets,capsules, sprays, lozenges, treated substrates (e.g., oral or topicalswabs, pads, or disposable, non-digestible substrate treated with thecompositions of the present invention); oral liquids (e.g., suspensions,solutions, emulsions), powders, suppositories, or any other suitabledosage form. In some embodiments, the present disclosure provides amethod for the manufacture of a pharmaceutical composition. Exemplarymethods include: contacting the microorganism (e.g., the non-pathogenicbacterium) with a pharmaceutically acceptable carrier, thereby formingthe pharmaceutical composition. In some examples, the method furtherincludes: growing the microorganism in a medium. The method may furtherinclude drying (e.g., freeze-drying) a liquid containing themicroorganism, wherein the liquid optionally includes thepharmaceutically acceptable carrier.

Unit Dosage Forms

The current disclosure further provides unit dosage forms comprising acertain amount of a microorganism (e.g., bacterium) of the presentdisclosure optionally in combination with a food-grade orpharmaceutically acceptable carrier. Exemplary unit dosage forms containfrom about 1×10³ to about 1×10¹⁴ colony-forming units (cfu) of anon-pathogenic microorganism (e.g., a non-pathogenic Gram-positivebacterium). Other exemplary unit dosage forms contain from about 1×10⁴to about 1×10′¹³ colony-forming units (cfu) of a non-pathogenicmicroorganism (e.g., a non-pathogenic Gram-positive bacterium), or fromabout 1×10⁴ to about 1×10¹² colony-forming units (cfu) of anon-pathogenic microorganism (e.g., a non-pathogenic Gram-positivebacterium). In other embodiments, the unit dosage form comprises fromabout 1×10S to about 1×10¹² colony-forming units (cfu), or from about1×10⁶ to about 1×10¹² colony-forming units (cfu) of the non-pathogenicmicroorganism (e.g., the non-pathogenic Gram-positive bacterium). Inother embodiments, the unit dosage form comprises from about 1×10⁵ toabout 1×10¹² colony-forming units (cfu), or from about 1×10⁹ to about1×10¹² colony-forming units (cfu) of the non-pathogenic microorganism(e.g., the non-pathogenic Gram-positive bacterium). In yet otherembodiments, the unit dosage form comprises from about 1×10⁹ to about1×10¹¹ colony-forming units (cfu), or from about 1×10⁹ to about 1×10¹⁰colony-forming units (cfu) of the non-pathogenic microorganism (e.g.,the non-pathogenic Gram-positive bacterium). In yet other embodiments,the unit dosage form comprises from about 1×10¹⁰ to about 1×10¹¹colony-forming units (cfu), or from about 1×10⁸ to about 1×10¹⁰colony-forming units (cfu) of the non-pathogenic microorganism (e.g.,the non-pathogenic Gram-positive bacterium).

In yet other embodiments, the unit dosage form comprises from about1×10⁹ to about 1×10¹⁰ colony-forming units (cfu), or from about 1×10 toabout 100×10⁹ colony-forming units (cfu) of the non-pathogenicmicroorganism (e.g., the non-pathogenic Gram-positive bacterium).

The unit dosage form can have any physical form or shape. In someembodiments, the unit dosage form is adapted for oral administration. Insome examples according to these embodiments, the unit dosage form is inthe form of a capsule, a tablet, or a granule. Exemplary capsulesinclude capsules filled with micro-granules. In some embodiments, thenon-pathogenic microorganism (e.g., the non-pathogenic Gram-positivebacterium) contained in the dosage form is in a dry-powder form. Forexample, the microorganism is in a freeze-dried powder form, which isoptionally compacted and coated.

This invention will be better understood by reference to the Examplesthat follow, but those skilled in the art will readily appreciate thatthese are only illustrative of the invention as described more fully inthe claims that follow thereafter. Additionally, throughout thisapplication, various publications are cited. The disclosures of thesepublications are hereby incorporated by reference into this applicationin their entirety to describe more fully the state of the art to whichthis invention pertains.

EXAMPLES Example 1 Generation of Lactococcus lactis Strains ExpressingCmbA on the Bacterial Surface

(a) Episomal Expression of CmbA

The codon usage of the CmbA encoding gene (see, SEQ ID NO: 2 and codingregion for CmbA in SEQ ID NO: 11, FIGS. 5A-D) was optimized to L. lactisMG1363. The resulting DNA sequence was made synthetically. Using overlayPCR assembly, PthyA>>SSusp45 (usp45 secretion signal) and cmbA werefused to form one PCR fragment. The PthyA>>SSusp45-cmbA expression unitwas positioned between the 5′thyA and 3′thyA regions of L. lactis strainMG1363 by PCR. The resulting PCR fragment:5′thyA>>PthyA>>SSusp45-cmbA>>3′thyA was subcloned into plasmid pORI19(see, e.g., Law et al., J. Bacteriol. 1995, 177(24):7011-7018) togenerate plasmid pAGX1893. Plasmid pAGX1893 was transformed intoLactococcus lactis strain LL108 resulting in strain LL108[pAGX1893].

(b) Chromosomal Integration (Constitutive Expression of CmbA)

Integration vector pAGX1893 was electroporated into two different L.lactis strains containing a bicistronic expression cassette containing aheterologous nucleic acid sequence encoding the therapeutic polypeptidePAL (gapB>>rpmD>>pal) (SEQ ID NOs: 24 and 25). These two strains weresAGX0599 and sAGX0585 (a trehalose accumulating strain). Insertion ofSSusp45-cmbA by homologous recombination (double-crossover) in the thyAlocus was confirmed by PCR and Sanger DNA sequencing. The resultingstrains were termed sAGX0618 and sAGX0619, respectively.

Example 2 Generation of Lactococcus lactis Strains Expressing MbpL onthe Bacterial Surface

(a) Episomal Expression of MbpL (Using Native Signal Peptide)

Construction of a [PthyA>>mbpL] thyA integration/expression vectorpAGX1903: The codon usage of the MbpL encoding gene (with signalpeptide; SSmbpL) was optimized to L. lactis MG1363. The resulting DNAsequence was made synthetically. Using overlay PCR assembly, PthyA andmbpL were fused to form one PCR fragment. The PthyA>>SSmbpL>>mbpLexpression unit (SEQ ID NO: 17) was positioned between the 5′thyA and3′thyA regions of L. lactis strain MG1363 by PCR. The resulting PCRfragment: 5′thyA>>PthyA>>SSmbpL>>mbpL>>3′thyA was subcloned into plasmidpORI19 to generate plasmid pAGX1903, which was transformed into L.lactis strain LL108 resulting in strain LL108[pAGX1903].

(b) Episomal Expression of MbpL (Using SSusp4S)

Another construct, which incorporates mbpL may include secretion leadersequence SSusp45 instead of MbpL's own signal peptide. A[PthyA>>SSusp45-mbpL] thyA integration/expression vector can be preparedin accordance with the procedure outlined in Example 1, wherein thenucleic acid sequence encoding cmbA can be replaced with a nucleic acidsequence encoding MbpL polypeptide without its own signal peptide. TheMbpL encoding gene (without signal peptide) can be optimized to L.lactis MG1363. The resulting DNA sequence can be made synthetically.Using overlay PCR assembly, PthyA>>SSusp45 and mbpL can be fused to formone PCR fragment. The PthyA>>SSusp45-mbpL expression unit (SEQ ID NO:15) can be positioned between the 5′thyA and 3′thyA regions of L. lactisstrain MG1363 by PCR. The resulting PCR fragment:5′thyA>>PthyA>>SSusp45-mbpL>>3′thyA can be subcloned, e.g., into plasmidpORI19 to generate another plasmid, which can be transformed into a L.lactis, e.g., strain LL108.

Example 3 Generation of Lactococcus lactis Strains Expressing MapA onthe Bacterial Surface

(a) Episomal Expression of MapA (Using Native Signal Peptide)

Construction of a [PthyA>>mapA] thyA integration/expression vectorpAGX1946: The mapA encoding gene (with signal peptide) was optimized toL. lactis MG1363. The resulting DNA sequence was made synthetically.Using overlay PCR assembly, PthyA and mapA were fused to form one PCRfragment. The PthyA SSmapA>>mapA expression unit (SEQ ID NO: 21) waspositioned between the 5′thyA and 3′thyA regions of L. lactis strainMG1363 by PCR. The resulting PCR fragment:5′thyA>>PthyA>>SSmapA>>mapA>>3′thyA was subcloned in plasmid pORI119 togenerate plasmid pAGX1946, which was transformed into L. lactis strainLL108 resulting in strain LL108[pAGX1946].

(b) Episomal Expression of MapA (Using SSusp4S)

Another construct, which incorporates mapA may include secretion leadersequence SSusp45 instead of MapA's own signal peptide. A[PthyA>>SSusp45>>mapA]thyA integration/expression vector can beprepared, e.g., in accordance with the procedure in Example 1, whereinthe nucleic acid sequence encoding cmbA can be replaced with a nucleicacid sequence encoding MapA polypeptide without its signal peptide. ThemapA encoding gene can be optimized to L. lactis MG1363. The resultingDNA sequence can be made synthetically. Using overlay PCR assembly,PthyA>>SSusp45 and mapA (without signal peptide) can be fused to formone PCR fragment. The PthyA>>SSusp45-mapA expression unit (SEQ ID NO:19) can be positioned between the 5′thyA and 3′thyA regions of L. lactisstrain MG1363 by PCR. The resulting PCR fragment:5′thyA>>PthyA>>SSusp45-mapA>>3′thyA can be subcloned, e.g., into plasmidpORI19 to generate another plasmid, which can be transformed into a L.lactis strain, e.g., LL108.

Example 4 Lactococcus lactis Strains Expressing an hTFF3-CmbA FusionProtein on the Bacterial Surface

(a) Expression of hTFF3

FIG. 4 illustrates an exemplary SSusp45-htff3 construct (amino acid andnucleic acid sequences) which can be used as the N-terminal portion of afusion protein, with a C-terminal adherence polypeptide. The C-terminaladherence polypeptide can be added through PCR fusion or moretraditional ligation.

(b) Episomal Expression of hTFF3-CmbA

A [PthyA>>SSusp45-hTFF3-cmbA] thyA integration/expression vector wasconstructed. The codon usage of the hTFF3 and CmbA (without signalpeptide) encoding genes were optimized to L. lactis MG1363. Theresulting htff3 and cmbA DNA sequences were made synthetically. Usingoverlay PCR assembly, PthyA>>SSusp45, hTFF3 and cmbA were fused to formone PCR fragment. The PthyA>>SSusp45-hTFF3-cmbA expression unit (see SEQID NO: 11 and FIGS. 5A-D) was positioned between the 5′thyA and 3′thyAregions of L. lactis strain MG1363 by PCR. The resulting PCR fragment:5′thyA>>PthyA>>SSup45-hTFF3-cmbA>>3′thyA was subcloned in plasmid pORI19(Law et al., 1995 supra) to generate plasmid pAGX2005 (see FIG. 1),which was transformed into L. lactis strain LL108 resulting in strainLL108[pAGX2005].

(b) Chromosomal Integration (Constitutive Expression of hTFF3-cmbA; thyAPromoter)

Integration vector pAGX2005 (see above and FIG. 1) was electroporatedinto two different thyA-wild type Lactococcus lactis strains, L. lactissAGX0599 and sAGX0585 (trehalose accumulating strain) containing abicistronic expression cassette encoding the therapeutic polypeptide PAL(gapB>>rpmD>>pal). Insertion of SSusp45-hTFF3-CmbA (see, e.g., SEQ IDNO: 11 and FIGS. 5A-D) by homologous recombination (double crossover) inthe thyA locus was confirmed by PCR and Sanger DNA sequencing. Theresulting strains were termed sAGX0644 and sAGX0645.

(c) Chromosomal Integration (Constitutive Expression of hTFF3-CmbA;PhllA Promoter)

Promoter replacement vector pAGX2041 (PthyA->PhllA) was electroporatedinto L. lactis strain sAGX0644 (see above) containing a bicistronicexpression cassette encoding the therapeutic polypeptide PAL(gapB>>rpmD>>pal) and PthyA>>SSusp45-hTFF3-cmbA. Replacement of PthyA byPhllA by homologous recombination (double crossover) was confirmed byPCR and Sanger DNA sequencing. The resulting strain was termed:sAGX0660.

Example 5 Lactococcus lactis Strains Expressing hTFF1-SpaX on theBacterial Surface

(a) Episomal Expression of h TFF1-spaX

Construction of a [PthyA>>SSusp45-hTFF1-spaX] (SEQ ID NOs: 22 and 23)thyA integration/expression vector pAGX1894: The codon usage of thehTFF1 encoding gene was optimized to L. lactis MG1363. The resulting DNAsequence was made synthetic. Using overlay PCR assembly, PthyA>>SSusp45,hTFF1 and the gene encoding for the cell wall anchor of protein A ofStaphylococcus aureus (SpaX, Steidler et al., 1998) were fused to formone PCR fragment. The PthyA>>SSusp45-hTFF1-spaX expression unit waspositioned between the 5′thyA and 3′thyA regions of L. lactis strainMG1363 by PCR. The resulting PCR fragment:5′thyA>>PthyA>>SSusp45-hTFF1-spaX>>3′thyA was subcloned into plasmidpORI19 to generate plasmid pAGX1894, which was transformed into L.lactis strain LL08 resulting in strain LL108[pAGX1894].

Example 6 Lactococcus lactis Strains Episomally Expressing Mucin-Bindingand Cell-Binding Polypeptides

The following plasmids and Lactococcus lactis strains were prepared asdescribed herein and were tested for their mucin- and cell-binding(Caco-2, IEC-18, and HT29-MTX cells) capabilities. The plasmids listedin Table 1 below were electroporated into L. lactis strain LL108.

TABLE 1 Lactococcus lactis Strains Episomally Expressing Mucin-Bindingand Cell-Binding Polypeptides BioAdhesion Mucin Cell Plasmid StrainConstruct binding binding pAGX1417 LL108[pAGX1417] none (control) − −pAGX1893 LL108[pAGX1893] PthyA >> cmbA − +++ pAGX1903 LL108[pAGX1903]PthyA >> mbpL − +++ pAGX1946 LL108[pAGX1946] PthyA >> mapA − + pAGX1894LL108[pAGX1894] PthyA >> +++ − SSusp45-hTFF1- spaX pAGX1986LL108[pAGX1986] PthyA >> N/A N/A SSusp45-hTFF3- spaX pAGX2005LL108[pAGX2005] PthyA >> +++ +++ SSusp45-hTFF3- cmbA (+):; (++):; (+++):cell recovery between 0 and 12% (indicates relative overall performancebased on various experiments, using various kinds of cells (Caco-2,IEC-18, HT29-MTX) and substrates (mucins type II & III).(a) Binding of Lactococcus Lactis LL108[pAGX2005] to Mucins

Experimental procedure: mucins (Sigma type II, cat # M2378-100G, mucinsform porcine stomach and Sigma type III, cat # M1778-10G, mucin fromporcine stomach, bound sialic acid (0.5-1.5%), partially purified) werecoated at 500 μg/ml in 50 mM carbonate buffer on Nunc MaxiSorp® plates.Plates were washed 3 times with PBS, blocked with PBS+Tween20 and washed3 times with PBS. Overnight saturated L. lactis cultures were diluted inPBS to OD₆₀₀=1. Cultures were washed with PBS+0.05% Tween20 andresuspended in 1 volume PBS+0.05% Tween20. 100 μl bacterial suspensionwas applied on each well. Plates were incubated for 16 hours at 4° C.Plates were washed 3 times with PBS+0.05% Tween20. Plates were dried for1 hour at 55° C. (A) OD was measured at 405 nm. (B) 100 μl per wellcrystal violet (1 mg/ml) was added and incubated for 45 minutes at roomtemperature. OD was subsequently measured at 595 nm.

Result:

FIG. 2 shows that strain LL108[pAGX894], expressing human trefoil factor1 (hTFF1) on its surface by use of the Staphylococcus aureus protein Aanchor fragment SpaX (Steidler et al., 1998, supra) and strainLL108[pAGX2005], expressing hTFF3-CmbA on its surface bind to mucinswith comparable strength (Sigma type II & type III) when measured usingtwo different readout methods. In contrast, a control strain containingan empty control vector, LL108[pAGX417] did not show any binding tomucins for the two readout methods.

(b) Adherence of Lactococcus Lactis LL108[pAGX2005] to Caco-2 Cells

Experimental procedure: Caco-2 cells were seeded in a 12-well plate at5×10⁴ cells/cm². Cells were grown for 17 days to differentiate.Overnight saturated L. lactis cultures were diluted 1/1000 in DMEM andwashed 3 times with DMEM. L. lactis cells were finally resuspended inDMEM. 1 ml of the bacterial suspensions was applied on the cells in the12-well plate and incubated for 1 hour at 37° C. Cells were washed 3times with DMEM. Cells were harvested by lysis with Triton-X100 (1 ml0.1% Triton-X100 in PBS was applied on the cells, incubation for 5 to 10minutes until total detachment of the cells). Harvested cell suspensionswere appropriately diluted and plated out on GM17E plates. Bacterialcounts on the plates were measured after 24 hours of incubation at 30°C. Percent recovery was determined as L. lactis cells that wererecovered from the plating of the harvested cell suspensions dividedwith the number of cells that were applied initially to the Caco-2cells.

FIG. 3 shows that strain LL108[pAGX893] expressing CmbA on its surfaceand strain LL108[pAGX2005] expressing hTFF3-CmbA on its surface exhibitcomparable adhesion to Caco-2 cells. In contrast, control strainLL108[pAGX417] (control) and strain LL108[pAGX1894)] expressinghTFF1-SpaX on its surface did not exhibit enhanced adherence to Caco-2cells.

Conclusion

L. lactis strains expressing an hTFF3-CmbA fusion polypeptide on theirsurface exhibit enhanced in vitro binding to mucins and enhancedadherence to Caco-2 cells when compared to corresponding strains notexpressing an hTFF3-CmbA fusion polypeptide.

Example 7 Construction of Integration Plasmids Containing BicistronicExpression Units

The integration plasmids listed in Table 2 below were prepared asdescribed herein using a construct incorporating an intergenic region(e.g., the intergenic region preceding rplN) and can be tested for theirmucin- and cell-binding capabilities as described herein. The plasmidslisted in Table 2 can be electroporated into a host cell, such as L.lactis strain LL108 to generate bacteria expressing (e.g.,constitutively expressing) polypeptides encoded by these constructs.

TABLE 2 Integration Plasmids Containing Bicistronic Expression UnitsPlasmid BioAdhesion Construct (in indicated operon) pAGX1935 PhllA >>hllA >> rplN >> cmbA pAGX1938 Pusp45 >> usp45 >> rplN >> cmbA pAGX1997PhllA >> hllA >> rplN >> SSusp45-hTFF1-spaX pAGX1998 PhllA >> hllA >>rplN >> SSusp45-hTFF3-spaX pAGX2016 PhllA >> hllA >> rplN >>SSusp45-hTFF3-cmbA

Example 8 Lactococcus lactis Strains Containing Chromosomally IntegratedExpression Units

The following plasmids and Lactococcus lactis strains were prepared asdescribed herein and were tested for their mucin- and cell-bindingcapabilities as described in Example 6. Results are illustrated in FIGS.6 and 7. The plasmids listed in Table 3 below were integrated into a L.lactis host strain containing a chromosomally integrated PAL expressionunit (PgapB>>gapB>>rpmD>>pal). Some of the host strains further includedgenetic modifications leading to intracellular trehalose accumulation(*). Exemplary trehalose accumulating strains (i) lack functionaltrehalose-6-phosphate phosphorylase (ΔtrePP) and lack functionalcellobiose-specific PTS system IIc component (ΔptcC) genes; (ii) expressan E. coli trehalose-6-phosphate phosphatase (E. coli otsB), and (iii)express trehalose transporter genes (PTS genes). See, e.g., U.S. Pat.No. 9,200,249 and U.S. Patent Application Publication 2014/0234371, thedisclosures of which are incorporated herein in their entireties.

Selected strains were also tested for PAL expression. Results(summarized in FIG. 8) indicate that expression of the cell adherenceand mucoadhesive polypeptides did not have a significant influence onthe amount of PAL expressed per cell (μg/10⁹ cells).

TABLE 3 Lactococcus lactis Strains Constitutively ExpressingMucin-Binding and Cell-Binding Polypeptides BioAdhesion Construct (thyAMucin Cell Strain Trehalose locus) binding binding sAGX0585 (*) none(control) − − sAGX0599 wt none (control) − − sAGX0618 wt PthyA >> cmbA −++ sAGX0619 (*) PthyA >> cmbA − ++ sAGX0644 wt PthyA >> ++ ++SSusp45-hTFF3-cmbA sAGX0645 (*) PthyA >> ++ ++ SSusp45-hTFF3-cmbAsAGX0660 wt PhllA >> +++ ++ SSusp45-hTFF3-cmbA (*) PhllA >> PTS; ΔtrePP; Δ ptcC; usp45 >> otsB. wt: wild-type (no trehalose accumulation)(+); (++); (+++): cell recovery between 0 and 12% (indicates relativeoverall performance based on various experiments, using various kinds ofcells (Caco-2, IEC-18, HT29-MTX) and substrates (mucins type II & III).

Example 9 Construction of Lactococcus lactis Strains ContainingChromosomally Integrated Expression Units

The Lactococcus lactis strains listed in Table 4 below can be preparedas described herein using appropriate constructs and can be tested fortheir mucin- and cell-binding capabilities as described herein

Each of these bacterial strains expressing mucin-binding andcell-adherence polypeptides on their surface (e.g., Lactococcus lactisstrains expressing hTFF3-CmbA fusion proteins on their surface) can beevaluated for their gastro-intestinal (GI)-transit time, and can becompared with corresponding strains not expressing the mucin-binding andcell-adherence polypeptides (e.g., corresponding strains not expressinga hTFF3-CmbA fusion polypeptide).

For example, an equal number of bacterial cells (e.g., based on cfu) ofthe bacterial strains, which either express hTFF3-CmbA, or do notexpress the fusion protein, can be administered to an individual (humanor animal). Non-episomal strains+/−hTFF3-CmbA can be equipped with anantibiotic selection marker (e.g., pT1NX, conveying erythromycinresistance). Episomal strains can be further selected based on their Emresistance. GI transit times may be measured by one of the followingmethods:

TABLE 4 Lactococcus lactis Strains Constitutively ExpressingMucin-Binding and Cell-Binding Polypeptides Strain Trehalose BioAdhesionConstruct (thyA locus) sAGX0620 wt PthyA >> SSmbpL >> mbpL sAGX0621 (a)PthyA >> SSmbpL >> mbpL sAGX0624 wt PthyA >> SSmapA >> mapA sAGX0625 (a)PthyA >> SSmapA >> mapA sAGX0661 (a) PhllA >> SSusp45-hTFF3-cmbA (*)PhllA >> PTS; Δ trePP; ΔptcC; usp45 >> otsB. wt: wild-type (no trehaloseaccumulation)

(1) At regular, (e.g., 1 h) intervals, total feces can be collected andthe number of cfu recovered of can be determined for each strain, e.g.,by Q-PCR or dilutive plating on solid agar containing erythromycin. Thiswill yield for every strain a kinetic of recovery, which can show a timepoint of collection where the number of cfu recovered is maximal. It isobserved that at time points beyond this maximum, more cfus arerecovered for hTFF3-CmbA+strains.

(2) At regular (e.g., 1 h) intervals, test animals (e.g., mice) can besacrificed, and the number of bacterial cfu recovered from those animalscan be determined by dilutive plating on solid agar containingerythromycin. This shows that at progressing time, more cfus arerecovered for hTFF3-CmbA+strains.

Example 10 Treatment of PKU—Gastrointestinal Degradation ofPhenylalanine Using PAL Expressed by Genetically Modified Bacteria ofthe Current Disclosure

The enzyme phenylalanine ammonia lyase (PAL), which converts Phe intothe cinnamic acid and does not require a co-factor, has been used totreat phenylketonuria (PKU). See, e.g., Sarkissian, C. N. et al., Proc.Natl. Acad. Sci. USA 2008, 105: 20894-20899. Because PAL is rapidlydegraded in the gastrointestinal (GI) tract, oral delivery is difficultto accomplish. A proof-of-concept study showed that oral administrationof PAL expressed by genetically engineered E. coli in PKU (enu2/2) miceresulted in a significant reduction in plasma Phe levels. See, e.g.,Sarkissian, C. N. et al., Proc. Natl. Acad. Sci. USA 1999, 96:2339-2344.However, safety concerns associated with the administration of E. coliprevents the use of this strategy in humans. A Lactococcus lactis strainexpressing PAL has been generated and evaluated in a PKU mouse model.Oral administration of the recombinant bacteria resulted in a reductionof deuterated Phe absorption. See, e.g., International PatentApplication Publication WO 2014066945.

To allow for the administration of genetically modified bacteriaexpressing PAL to human PKU patients, the inventors have prepared“clinical grade” strains, which constitutively express the PAL enzyme,and are further modified to exhibit increased GI transit times, greaterstability under GI conditions, and self-containment. Chromosomalintegration of the bioactive polypeptide is important because episomalexpression is associated with safety concerns. For example, episomes canbe readily transmitted to other bacteria in the gastro-intestinalsystem. Further, episomal maintenance can provide hurdles formanufacturing. Additionally, known PAL producing strains are rapidlydestroyed in the proximal GI tract. Further, there is a concern that theknown PAL producing bacteria could survive and propagate outside of thebody.

Multiple L. lactis strains with one or more of the following improvedfeatures were generated, e.g., as described herein above in Example8: 1) a PAL expression cassette that is integrated into the bacterialchromosome and driven by a constitutive promoter for improved safety andease of manufacture; 2) modifications that promote trehaloseaccumulation, which improves L. lactis survival in the GI tract (see,e.g., Termont, S. et al., Appl. Environ. Microbiol. 2006, 72:7694-7700); 3) incorporation of an auxotrophic dependency on thymidineto prevent survival outside the human body (see, e.g., Steidler, L. etal., Nat. Biotechnol. 2003, 21: 785-789); and 4) genetic modificationsto express mucoadhesive surface proteins to improve retention in theproximal GI tract and prolong Phe degradation (see, e.g., Caluwaerts, S.et al., Oral. Oncol. 2010, 46:564-570). The strains were evaluated forPAL expression and their respective cell adherence and mucoadhesiveproperties as described in Examples 6 and 8. Surface expression of theadhesion protein improved bacterial retention on Caco-2 monolayers byapproximately 8-fold. Adhesion to both Type II and Type III mucins wassimilarly enhanced. Importantly, the trehalose modification for improvedsurvival in the GI tract and the expression of the adhesion molecule didnot alter PAL levels expressed by the bacteria.

Selected bacterial strains, e.g., Lactococcus lactis strains (e.g.,strains constitutively expressing PAL; e.g., sAGX0599, sAGX0644,aAGX0585, and sAGX0645) can be tested for their efficacy in the enu2/2“PKU” mouse model to identify strains suitable for human clinicaltrials.

In the first study, sAGX0599, and sAGX0645 were tested in the PKU micethat received a dose of deuterated Phe concurrently with 10⁹ cfu of L.lactis via gavage. Blood was sampled at 15 minute intervals for one hourand assayed for deuterated Phe. Positive control animals received aknown episomal recombinant strain, e.g., as described in InternationalPatent Application Publication WO 2014066945). Negative control animalsreceived no bacteria. As shown in FIG. 9, the total Phe absorbed by micewas lowest in mice fed sAGX0599 (secreting PAL) and sAGX0645 (PAL andTFF3-CmbA), and was superior to the positive control. Interestingly, thefinal level of Phe was lowest in the mice fed sAGX0645 (PAL andTFF3-CmbA), but the Cmax of Phe was higher. This may reflect thatsAGX0645 is moving slower down the small bowel towards the particularsite of Phe uptake compared to L. lactis without the adhesion fusionprotein. A lower Cmax and AUC may be obtained by administration of thePhe-degrading bacteria can prior to feeding. A lower Cmax and AUC mayalso be obtained by regular administration of the bacteria with a mealsuch that distal Phe-degrading bacteria to reduce Phe from one meal arealready present from a previous meal.

It should be noted that feeding Phe is an artificial model of PKU.Single amino acids are very efficiently absorbed. In typical situations,proteins are broken down as they pass through the intestine such thatfree amino acids available for absorption occur primarily in more distalportions than the upper small intestine. Thus, a PAL-secreting bacteriumlocated in the more distal portion of the intestine may be able tobetter block Phe uptake from food than as suggested by experimentsadministering Phe directly.

Moreover, L. lactis may be modified with different cell and mucusadhesion molecules to localize the delivery of PAL to the optimalsection(s) of the intestine.

A second study may assess the pharmacodynamics of the selected bacterialstrains, e.g., Lactococcus lactis strains. Bacteria can be administeredup to six hours before the deuterated Phe. Blood can be assayed asdescribed above. The resulting data can inform the scheduling forsubsequent studies and provide information relevant to clinicalapplication. For example, humans may take one unit dosage form (e.g.,capsule) containing the bacteria with each meal.

A third study may assess the long-term effectiveness of the bacteria inlowering Phe blood levels. Animals can be gavaged with a selectedbacterial strain (e.g., Lactococcus lactis strain), e.g., optimal strainfrom the first study, and can then be fed a standard chow diet. Forexample, two L. lactis-associated feedings each day with the durationfor each feeding determined by the outcome from the second. Blood Phelevels can be assessed regularly, e.g., semi-weekly for three weeks.Controls can be those employed in the first study, and can also includea low Phe diet cohort. Note: this study may not use deuterated Phe. Apositive outcome can be a statistically significant improvement in Pheblood levels, e.g., sufficient to likely result in clinical efficacy.Other studies can combine bacterial therapy (e.g., L. lactis therapy)with dietary intervention.

Each study can utilize cohorts of 5 mice to provide sufficient power forstatistical analyses, and mice can be reused after a washout. Phe can beassayed by tandem mass spectrometry, which is both sensitive andspecific and requires minimal amounts of blood.

Example 11 Treatment of Oral Mucositis

Oral mucositis is a breakdown of the oral mucosa and is a commoncomplication of cancer therapy, especially for treatment of head andneck cancer. TFF1 is secreted in the upper GI tract and is associatedwith protection and healing of mucosal surfaces. TFF1 shows promise as atreatment for oral mucositis. To increase delivery of TFF1 at the oralmucosa, L. lactis is engineered to express both an hTFF1-CmbA fusionalong with “free” hTFF1, both on the chromosome. If this was done stepwise, there would be risk that one htff1 gene would recombine withanother htff1 gene on the chromosome. To minimize this risk, hTFF1-cmbAand htff1 can be constructed polycistronically, and are therefore ableto transform and integrate into the chromosome at one step. Further, thethird base in each codon can changed in one tff1 sequence so that itsnucleotide sequence differs from the other tff1 sequence, but thetranslated hTFF1 is the same. For example, there are six alanine codonswith the following frequency: UCU (18.6), UCC (4.0), UCA (20.6), UCG(3.9), AGU (16.7), and AGC (7.3). Therefore, UCC and UCG may beinterchangeable. UCU, UCA, and to a lesser extent AGU, may beinterchangeable.

After modifying the codon frequency, thePhllA>>SSusp45>>hTFF1>>rpmD>>SSusp45>>hTFF1-CmbA is constructed throughPCR, and is cloned between the 5′ thyA and 3′ thyA on a conditionallyreplicative carrier plasmid derived from pORI19. Transformation into L.lactis and selection, as described elsewhere herein, lead to integrationinto chromosomal thyA locus.

The resulting strain is shown to secrete TFF1 and express a TFF-CmbAfusion that mediates increased binding to oral mucus compared with astrain expressing TFF1 alone. The strain is tested in models ofmucositis, and the presence of the TFF-CmbA increases persistence in theoral cavity and increases delivery of therapeutic TFF1, such that thefrequency of dosing may be decreased.

Example 12 Treatment of Diabetes

The Lactococcus lactis strain secreting hTFF3-CmbA, as described above,is modified to express human proinsulin PINS and human IL10. Forexample, a construct for hPINS expression may be hPINS with a SSUsp45secretion leader, under the control of the strong gapB promoter andafter the gapB gene and rpmD spacer (i.e.,PgapB>>gapB>>rpmD>>usp45>>PINS). For hIL-10, a construct may bePhllA>>SSusp45>>hil-10.

Because hTFF3-CmbA are already inserted into the thyA locus, PINS andIL-10 may be inserted into another genomic site, such as ptcC or trePP.PtcC and trePP mutations are associated with trehalose accumulation. Theresulting strain is tested for expression of hIL-10, PINS andhTFF3-CmbA. The strain is then tested in NOD mice, an animal model oftype 1 diabetes, against a strain lacking hTFF3-CmbA. The strain lackinghTFF3-CmbA has previously been shown to generate a Treg response toPINS, reverse the autoimmune response to beta cells of the pancreas, andthereby treat diabetes. The treatment is especially efficacious inrecent onset disease. The presence of the hTFF-CmbA may increase gutcolonization and persistence, resulting in a stronger Treg response.

While some embodiments have been shown and described herein, suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the invention. It should be understood thatvarious alternatives to the embodiments of the invention describedherein will be employed in practicing the invention.

Exemplary Embodiments

-   1. A bacterium comprising an exogenous nucleic acid encoding a    fusion protein, wherein said fusion protein comprises a    cell-adherence polypeptide.-   2. The bacterium of embodiment 1, wherein said cell-adherence    polypeptide is selected from the group consisting of cell and    mucus-binding protein A (CmbA), mucus binding protein (Mub), mucus    adhesion promoting protein (MapA), lactococcal mucin binding protein    (MbpL), and any combination thereof.-   3. The bacterium of embodiment 2, wherein said cell-adherence    polypeptide is CmbA.-   4. The bacterium of embodiment 3, wherein said CmbA is Lactobacillus    reuteri CmbA.-   5. The bacterium of any one of the preceding embodiments, wherein    said fusion protein further comprises a mucin-binding polypeptide.-   6. The bacterium of embodiment 5, wherein said mucin-binding    polypeptide is a trefoil factor (TFF) polypeptide.-   7. The bacterium according to embodiment 6, wherein said fusion    protein comprises CmbA and a TFF.-   8. The bacterium of any one of embodiments 1 to 7, wherein said    exogenous nucleic acid encoding a fusion protein is integrated into    the chromosome of said bacterium.-   9. The bacterium of any one of embodiments 1 to 7, wherein said    exogenous nucleic acid encoding a fusion protein is located on a    plasmid.-   10. The bacterium of any one of the preceding embodiments, wherein    said fusion protein is expressed by said bacterium.-   11. The bacterium of embodiment 10, wherein said fusion protein is    anchored in a cell wall of said bacterium.-   12. The bacterium of any one of the preceding embodiments, wherein    said exogenous nucleic acid encoding a fusion protein further    comprises a secretion leader sequence encoding a secretion signal    peptide.-   13. The bacterium of embodiment 12, wherein said secretion leader    sequence is SSusp45.-   14. The bacterium of embodiment 13, wherein said secretion leader    sequence encodes a secretion signal peptide having an amino acid    sequence that is at least 90% identical to SEQ ID NO: 5.-   15. The bacterium of any one of embodiments 12 to 14, wherein said    secretion signal peptide is bound to said mucin-binding polypeptide.-   16. The bacterium of any one of embodiments 12 to 15, wherein said    secretion signal peptide is bound to a linker.-   17. The bacterium of any one of embodiments 12 to 16, wherein said    secretion signal peptide is cleaved from said fusion protein.-   18. The bacterium of embodiment 17, wherein said secretion signal    peptide is cleaved from said fusion protein when said fusion protein    is anchored in a cell wall of said bacterium.-   19. The bacterium of any one of embodiments 1 to 18, wherein said    exogenous nucleic acid encoding a fusion protein is    transcriptionally regulated by a promoter selected from the group    consisting of a thyA promoter (PthyA), an hlla promoter (Phi/A), and    a gapB promoter.-   20. The bacterium of embodiment 19, wherein said exogenous nucleic    acid encoding a fusion protein is transcriptionally regulated by    PthyA.-   21. The bacterium of embodiment 19, wherein said exogenous nucleic    acid encoding a fusion protein is transcriptionally regulated by    PhllA.-   22. A bacterium comprising a fusion protein anchored in a cell-wall    of said bacterium, wherein said fusion protein comprises a TFF    polypeptide and a CmbA polypeptide.-   23. The bacterium of any one of embodiments 1 to 22, wherein said    bacterium is a Gram-positive bacterium.-   24. The bacterium of embodiment 23, wherein said Gram-positive    bacterium is non-pathogenic.-   25. The bacterium of any one of embodiments 1 to 24, wherein said    bacterium is a lactic acid bacterium (LAB).-   26. The bacterium of embodiment 25, wherein said LAB is selected    from the group consisting of a Lactococcus species (sp.) bacterium,    a Lactobacillus sp. bacterium, a Bifidobacterium sp. bacterium, a    Streptococcus sp. bacterium, and an Enterococcus sp. bacterium.-   27. The bacterium of embodiment 25, wherein said LAB is selected    from the group consisting of Lactococcus garvieae, Lactococcus    lactis, Lactococcus piscium, Lactococcus plantarum, Lactococcus    raffinolactis, Lactobacillus acetotolerans, Lactobacillus    acidophilus, Lactobacillus agilis, Lactobacillus algidus,    Lactobacillus alimentarius, Lactobacillus amylolyticus,    Lactobacillus amylophilus, Lactobacillus amylovorus, Lactobacillus    animalis, Lactobacillus aviarius, Lactobacillus aviarius subsp.    araffinosus, Lactobacillus aviarius subsp. aviarius, Lactobacillus    bavaricus, Lactobacillus bifermentans, Lactobacillus brevis,    Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus    carnis, Lactobacillus casei, Lactobacillus casei subsp. alactosus,    Lactobacillus casei subsp. casei, Lactobacillus casei subsp.    pseudoplantarum, Lactobacillus casei subsp. rhamnosus, Lactobacillus    casei subsp. tolerans, Lactobacillus catenaformis, Lactobacillus    cellobiosus, Lactobacillus collinoides, Lactobacillus confusus,    Lactobacillus coryniformis, Lactobacillus coryniformis subsp.    coryniformis, Lactobacillus coryniformis subsp. torquens,    Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus    curvatus subsp. curvatus, Lactobacillus curvatus subsp. melibiosus,    Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp.    bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii,    Lactobacillus delbrueckii subsp. lactis, Lactobacillus divergens,    Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus    fornicalis, Lactobacillus fructivorans, Lactobacillus fructosus,    Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus    graminis, Lactobacillus halotolerans, Lactobacillus hamsteri,    Lactobacillus helveticus, Lactobacillus heterohiochii. Lactobacillus    hilgardii, Lactobacillus homohiochii, Lactobacillus iners,    Lactobacillus intestinalis, Lactobacillus jensenii, Lactobacillus    johnsonii, Lactobacillus kandleri, Lactobacillus kefiri,    Lactobacillus kefiranofaciens, Lactobacillus kefirgranum,    Lactobacillus kunkeei, Lactobacillus lactis, Lactobacillus    leichmannii, Lactobacillus lindneri, Lactobacillus malefermentans,    Lactobacillus mali, Lactobacillus maltaromicus, Lactobacillus    manihotivorans, Lactobacillus minor, Lactobacillus minutus,    Lactobacillus mucosae, Lactobacillus murinus, Lactobacillus nagelii,    Lactobacillus oris, Lactobacillus panis, Lactobacillus parabuchneri,    Lactobacillus paracasei, Lactobacillus paracasei subsp. paracasei,    Lactobacillus paracasei subsp. tolerans, Lactobacillus parakefiri,    Lactobacillus paralimentarius, Lactobacillus paraplantarum,    Lactobacillus pentosus, Lactobacillus perolens, Lactobacillus    piscicola, Lactobacillus plantarum, Lactobacillus pontis,    Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rimae,    Lactobacillus rogosae, Lactobacillus ruminis, Lactobacillus sakei,    Lactobacillus sakei subsp. camosus, Lactobacillus sakei subsp.    sakei, Lactobacillus salivarius, Lactobacillus salivarius subsp.    salicinius, Lactobacillus salivarius subsp. salivarius,    Lactobacillus sanfranciscensis, Lactobacillus sharpeae,    Lactobacillus suebicus, Lactobacillus trichodes, Lactobacillus uli,    Lactobacillus vaccinostercus, Lactobacillus vaginalis, Lactobacillus    viridescens, Lactobacillus vitulinus, Lactobacillus xylosus,    Lactobacillus yamanashiensis, Lactobacillus yamanashiensis subsp.    mali, Lactobacillus yamanashiensis subsp. Yamanashiensis,    Lactobacillus zeae, Bifidobacterium adolescentis, Bifidobacterium    angulatum, Bifidobacterium bifidum, Bifidobacterium breve,    Bifidobacterium catenulatum, Bifidobacterium longum, Bifidobacterium    infantis, Enterococcus alcedinis, Enterococcus aquimarinus,    Enterococcus asini, Enterococcus avium, Enterococcus caccae,    Enterococcus camelliae, Enterococcus canintestini, Enterococcus    canis, Enterococcus casselifavus, Enterococcus cecorum, Enterococcus    columbae, Enterococcus devriesei, Enterococcus diestrammenae,    Enterococcus dispar, Enterococcus durans, Enterococcus eurekensis,    Enterococcus faecalis, Enterococcus faecium. Enterococcus    gallinarum, Enterococcus gilvus, Enterococcus haemoperoxidus,    Enterococcus hermanniensis, Enterococcus hirae, Enterococcus    italicus, Enterococcus lactis, Enterococcus lemanii, Enterococcus    malodoratus, Enterococcus moraviensis, Enterococcus mundtii,    Enterococcus olivae, Enterococcus pallens, Enterococcus    phoeniculicola, Enterococcus plantarum, Enterococcus pseudoavium,    Enterococcus quebecensis, Enterococcus raffinosus, Enterococcus    ratti, Enterococcus rivorum, Enterococcus rotai, Enterococcus    saccharolyticus, Enterococcus silesiacus, Enterococcus solitarius,    Enterococcus sulfureus, Enterococcus termitis, Enterococcus    thailandicus, Enterococcus ureasiticus, Enterococcus ureilyticus,    Enterococcus viikkiensis, Enterococcus villorum, Enterococcus    xiangfangensis, Streptococcus agalactiae, Streptococcus anginosus,    Streptococcus bovis, Streptococcus canis, Streptococcus    constellatus, Streptococcus dysgalactiae, Streptococcus equinus,    Streptococcus iniae, Streptococcus intermedius, Streptococcus    milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus    oralis, Streptococcus parasanguinis, Streptococcus peroris,    Streptococcus pneumoniae, Streptococcus pseudopneumoniae,    Streptococcus pyogenes, Streptococcus ratti, Streptococcus    salivarius, Streptococcus tigurinus, Streptococcus thermophilus,    Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis,    Streptococcus uberis, Streptococcus vestibularis, Streptococcus    viridans, and Streptococcus zooepidemicus.-   28. The bacterium of embodiment 27, wherein said bacterium is    Lactococcus lactis.-   29. The bacterium of embodiment 28, wherein said Lactococcus lactis    is selected from the group consisting of Lactococcus lactis subsp.    cremoris, Lactococcus lactis subsp. hordniae, and Lactococcus lactis    subsp. lactis.-   30. The bacterium of any one of embodiments 6 to 29, wherein said    TFF polypeptide is selected from the group consisting of TFF1, TFF2,    and TFF3.-   31. The bacterium of any one of embodiments 6 to 29, wherein said    TFF polypeptide is selected from the group consisting of human TFF,    mouse TFF, pig TFF, dog TFF, cat TFF, cow TFF, sheep TFF, fish TFF,    and amphibial TFF.-   32. The bacterium of embodiment 31, wherein said TFF polypeptide is    human TFF.-   33. The bacterium of embodiment 32, wherein said human TFF is human    TFF3.-   34. The bacterium of any one of embodiment 6 to 29, wherein said TFF    polypeptide has an amino acid sequence at least 80% identical to SEQ    ID NO: 3.-   35. The bacterium of any one of embodiments 6 to 29, wherein said    TFF polypeptide is a TFF variant polypeptide.-   36. The bacterium of embodiment 35, wherein said TFF variant    polypeptide has enhanced mucin-binding capability compared to a    corresponding wild-type TFF polypeptide.-   37. The bacterium of any one of embodiments 1 to 36, wherein said    CmbA polypeptide has an amino acid sequence at least 80% identical    to SEQ ID NO: 1.-   38. The bacterium of any one of embodiments 1 to 37, further    comprising an exogenous nucleic acid encoding at least one    therapeutic polypeptide.-   39. The bacterium of embodiment 38, wherein said at least one    therapeutic polypeptide is a cytokine.-   40. The bacterium of embodiment 38 or 39, wherein said at least one    therapeutic polypeptide is an interleukin (IL).-   41. The bacterium of embodiment 40, wherein said interleukin is    selected from the group consisting of IL-2, IL-10, IL-18, and any    combinations thereof.-   42. The bacterium of embodiment 38, wherein said at least one    therapeutic polypeptide is an antigen.-   43. The bacterium of embodiment 42, wherein said at least one    therapeutic polypeptide is an antigen and an interleukin.-   44. The bacterium of embodiment 43, wherein said interleukin is IL-2    or IL-10.-   45. The bacterium of any one of embodiments 42 to 44, wherein said    antigen is an autoantigen.-   46. The bacterium of embodiment 45, wherein said autoantigen is a    type-1 diabetes (T1D)-specific antigen.-   47. The bacterium of embodiment 46, wherein said T1D-specific    antigen is selected from the group consisting of proinsulin (PINS),    glutamic acid decarboxylase (GAD65), insulinoma-associated protein 2    (IA-2), islet-specific glucose-6-phosphatase catalytic    subunit-related protein (IGRP), zinc transporter 8 (ZnT8),    chromogranin A, (prepro) islet amyloid polypeptide (ppIAPP),    peripherin, citrullinated glucose-regulated protein (GRP), and any    combinations thereof.-   48. The bacterium of embodiment 38, wherein said at least one    therapeutic polypeptide is an antibody or a fragment thereof.-   49. The bacterium of embodiment 48, wherein said antibody is a    single-domain antibody or a nanobody.-   50. The bacterium of embodiment 48 or 49, wherein said antibody is    selected from the group consisting of an anti TNFα antibody, an    antibody to IL-4, an antibody to IL-5, an antibody to IL-13, an    antibody to IL-15, an antibody to immunoglobulin E (IgE), and any    combinations thereof.-   51. The bacterium of embodiment 48 or 49, wherein said antibody is a    fusion protein comprising a soluble TNF receptor and an Fc region of    an antibody.-   52. The bacterium of embodiment 38, wherein said at least one    therapeutic polypeptide is an enzyme or fragment thereof.-   53. The bacterium of embodiment 52, wherein said enzyme or fragment    thereof is functional.-   54. The bacterium of embodiment 52 or 53, wherein said enzyme or    fragment thereof is selected from the group consisting of a    phenylalanine ammonia lyase (PAL), an amino acid decarboxylase, and    any combinations thereof.-   55. The bacterium of embodiment 54, wherein said enzyme or fragment    thereof is PAL.-   56. The bacterium of embodiment 38, wherein said at least one    therapeutic polypeptide is selected from the group consisting of    glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2),    glucagon, exendin-4, and any combinations thereof.-   57. The bacterium of embodiment 38, wherein said at least one    therapeutic polypeptide is a growth factor.-   58. The bacterium of embodiment 57, wherein said growth factor is    epidermal growth factor (EGF).-   59. The bacterium of embodiment 58, wherein said EGF is human EGF or    porcine EGF.-   60. The bacterium of embodiment 38, wherein said at least one    therapeutic polypeptide is a TFF.-   61. The bacterium of embodiment 60, wherein said TFF is selected    from the group consisting of TFF1, TFF2, TFF3, and any combinations    thereof.-   62. The bacterium of any one of embodiments 38 to 61, wherein said    therapeutic polypeptide is a human polypeptide.-   63. The bacterium of any one of embodiments 42 to 44, wherein said    antigen is an allergen.-   64. The bacterium of embodiment 63, wherein said allergen is    selected from the group consisting of a tree pollen allergen, a weed    pollen allergen, a grass pollen allergen, a food allergen, a    dust-mite allergen, a mold allergen, an animal dander allergen, and    any combinations thereof.-   65. The bacterium of embodiment 64, wherein said allergen is a weed    pollen allergen.-   66. The bacterium of embodiment 65, wherein said weed pollen    allergen is a ragweed pollen allergen.-   67. The bacterium of embodiment 64, wherein said allergen is a tree    pollen allergen.-   68. The bacterium of embodiment 67, wherein said tree pollen    allergen is a birch pollen allergen or a Japanese cedar pollen    allergen.-   69. The bacterium of embodiment 64, wherein said allergen is a food    allergen.-   70. The bacterium of embodiment 69, wherein said food allergen is    selected from the group consisting of a peanut allergen, a milk    allergen, an egg allergen, a gluten allergen (gliadin epitope), and    any combinations thereof.-   71. The bacterium of any one of embodiments 38 to 70, wherein said    exogenous nucleic acid encoding at least one therapeutic polypeptide    is transcriptionally regulated by a gapB promoter.-   72. The bacterium of any one of embodiments 1 to 71, wherein said    bacterium has an increased gastro-intestinal (GI) transit time when    compared to a corresponding bacterium not comprising said exogenous    nucleic acid or not comprising said fusion protein.-   73. The bacterium of embodiment 72, wherein said GI transit time is    increased by at least about 10%.-   74. The bacterium of embodiment 73, wherein said GI transit time is    increased by at least about 30%.-   75. The bacterium of embodiment 72, wherein said GI transit time is    increased from at least about 10% to about 50%.-   76. The bacterium of any one of embodiments 1 to 75, wherein said    bacterium has increased in vitro mucin-binding capability when    compared to a corresponding bacterium not comprising said exogenous    nucleic acid or not comprising said fusion protein.-   77. The bacterium of embodiment 76, wherein said in vitro    mucin-binding capability is increased by at least about 20%.-   78. The bacterium of embodiment 77, wherein said in vitro    mucin-binding capability is increased by at least about 50%.-   79. The bacterium of embodiment 78, wherein said in vitro    mucin-binding capability is increased by at least about 100% (about    2×).-   80. The bacterium of embodiment 76, wherein said in vitro    mucin-binding capability is increased from at least about 20% to    about 500%.-   81. The bacterium of any one of embodiments 76 to 80, wherein said    in vitro mucin-binding capability is measured by binding said    bacterium to mucins from porcine stomach followed by:    -   (a) detecting light absorbance at 405 nm (OD₄₀₅); or    -   (b) staining said bacterium with crystal violet and subsequently        detecting light absorbance at 595 nm (OD₅₉₅).-   82. The bacterium of any one of embodiments 1 to 81, wherein said    bacterium has increased in vitro Caco-2 cell-binding capability when    compared to a corresponding bacterium not comprising said exogenous    nucleic acid or not comprising said fusion protein.-   83. The bacterium of embodiment 82, wherein said in vitro Caco-2    binding capability is increased by at least about 10%.-   84. The bacterium of embodiment 83, wherein said in vitro Caco-2    binding capability is increased by at least about 100% (about 2×).-   85. The bacterium of embodiment 84, wherein said in vitro Caco-2    binding capability is increased by at least about 400% (about 5×).-   86. The bacterium of embodiment 82, wherein said in vitro Caco-2    binding capability is increased from by at least about 10% to about    500%.-   87. The bacterium of any one of embodiments 82 to 86, wherein said    in vitro Caco-2 binding capability is measured by:    -   (a) contacting a culture of said bacterium with Caco-2 cells;    -   (b) washing said Caco-2 cells to remove unbound bacterial cells;    -   (c) detaching bacterial cells bound to said Caco-2 cells,        thereby forming detached bacterial cells; and    -   (d) titering said detached bacterial cells.-   88. The bacterium of any one of embodiments 1 to 87, wherein said    bacterium exhibits increased adherence to intestinal mucosa when    compared to a corresponding bacterium not comprising said exogenous    nucleic acid or not comprising said fusion protein.-   89. The bacterium of embodiment 88, wherein said adherence to    intestinal mucosa is increased from at least about 10% to about    500%.-   90. A composition comprising the bacterium of any one of embodiments    1 to 89.-   91. A pharmaceutical composition comprising the bacterium of any one    of embodiments 1 to 89, and a pharmaceutically acceptable carrier.-   92. The bacterium of any one of embodiments 1 to 89, the composition    of embodiment 90, or the pharmaceutical composition of embodiment    91, for use in the treatment or prevention of a disease selected    from the group consisting of an autoimmune disease, an allergy, a    metabolic disease, and a gastro-intestinal disease.-   93. The bacterium of any one of embodiments 1 to 89, the composition    of embodiment 90, or the pharmaceutical composition of embodiment    91, for use in the preparation of a medicament for the treatment or    prevention of a disease.-   94. The bacterium, the composition, or pharmaceutical composition of    embodiment 93, wherein said disease is selected from the group    consisting of an autoimmune disease, an allergy, a metabolic    disease, a gastro-intestinal disease, and a nutritional defect.-   95. A method for the treatment of a disease in a subject in need    thereof comprising administering to said subject a therapeutically    effective amount of the bacterium of any one of embodiments 1 to 89,    the composition of embodiment 90, or the pharmaceutical composition    of embodiment 91.-   96. The method of embodiment 95, wherein said disease is selected    from the group consisting of an autoimmune disease, an allergy, a    metabolic disease, a gastro-intestinal disease, and any combination    thereof.-   97. The method of embodiment 96, wherein said disease is an    autoimmune disease.-   98. The method of embodiment 97, wherein said autoimmune disease is    type-1 diabetes (T1D).-   99. The method of embodiment 96, wherein said disease is a metabolic    disease.-   100. The method of embodiment 99, wherein said metabolic disease is    phenylketonuria (PKU).-   101. The method of embodiment 96, wherein said disease is a    gastro-intestinal disease.-   102. The method of embodiment 101, wherein said gastro-intestinal    disease is celiac disease.-   103. The method of embodiment 101, wherein said gastro-intestinal    disease is inflammatory bowel disease (IBD).-   104. The method of embodiment 103, wherein said IBD is Crohn's    disease or ulcerative colitis.-   105. A method for preparing a genetically modified bacterium    comprising contacting a bacterium with an exogenous nucleic acid    encoding a fusion protein, wherein said exogenous nucleic acid    encoding a fusion protein comprises a sequence encoding a    cell-adherence polypeptide.-   106. The method of embodiment 105, wherein said contacting occurs    under conditions sufficient for said bacterium to internalize said    exogenous nucleic acid.-   107. The method of embodiment 105 or 106, wherein said exogenous    nucleic acid is located on a plasmid.-   108. The method of embodiment 106 or 107, wherein said exogenous    nucleic acid is integrated into a chromosome of said bacterium.-   109. The method of any one of embodiments 105 to 108, further    comprising culturing said bacterium and expressing said fusion    protein in said bacterium.-   110. The method of any one of embodiments 105 to 109, wherein said    cell-adherence polypeptide is selected from the group consisting of    cell and mucus-binding protein A (CmbA), Mub, MapA, MbpL, and any    combinations thereof.-   111. The method of embodiment 110, wherein said cell-adherence    polypeptide is CmbA.-   112. The method of embodiment 111, wherein said CmbA is    Lactobacillus reuteri CmbA.-   113. The method of any one of embodiments 105 to 112, wherein said    exogenous nucleic acid encoding a fusion protein further comprises a    sequence encoding a mucin-binding polypeptide.-   114. The method of embodiment 113, wherein said mucin-binding    polypeptide is a trefoil factor (TFF) polypeptide.-   115. The method of embodiment 114, wherein said exogenous nucleic    acid encoding a fusion protein comprises a sequence encoding CmbA    and a sequence encoding a TFF polypeptide.-   116. The method of any one of embodiments 105 to 115, wherein said    exogenous nucleic acid encoding a fusion protein is integrated into    the chromosome of said bacterium.-   117. The method of embodiment 116, wherein said exogenous nucleic    acid is integrated into the chromosome of said bacterium using    homologous recombination.-   118. The method of any one of embodiments 105 to 117, further    comprising contacting said bacterium with an exogenous nucleic acid    encoding a therapeutic polypeptide.-   119. The method of embodiment 118, wherein said contacting said    bacterium with an exogenous nucleic acid encoding a therapeutic    polypeptide occurs prior to said contacting said bacterium with an    exogenous nucleic acid encoding a fusion protein.-   120. The method of embodiment 118, wherein said contacting said    bacterium with an exogenous nucleic acid encoding a therapeutic    polypeptide occurs subsequent to said contacting said bacterium with    an exogenous nucleic acid encoding a fusion protein.-   121. The method of any one of embodiments 105 to 120, wherein said    genetically modified bacterium exhibits increased muco- and    cell-adhesive properties when compared to a corresponding bacterium    not comprising said exogenous nucleic acid encoding a fusion    protein.-   122. The method of any one of embodiments 105 to 121, further    comprising combining a culture of said genetically modified    bacterium with at least one cryopreserving agent to form a bacterial    mixture.-   123. The method of embodiment 122 further comprising freeze-drying    said bacterial mixture to form a freeze-dried composition.-   124. The method of any one of embodiments 105 to 121 or embodiment    123 further comprising combining said genetically modified    bacterium, or said freeze-dried composition with a pharmaceutically    acceptable carrier to form a pharmaceutical composition.-   125. The method of embodiment 123 or 124 further comprising    formulating said freeze-dried composition or said pharmaceutical    composition into a pharmaceutical dosage form.-   126. A genetically modified bacterium prepared by the method of any    one of embodiments 105 to 125.-   127. A unit dosage form comprising the bacterium of any one of    embodiments 1 to 89, the composition of embodiment 90, or the    pharmaceutical composition of embodiment 91.-   128. The unit dosage form of embodiment 127, wherein said unit    dosage form is an oral dosage form.-   129. The unit dosage form of embodiment 128, wherein said oral    dosage form is selected from the group consisting of a tablet, a    capsule, a sachet, and a packaged liquid.-   130. A method for enhancing growth in a mammal comprising    administering to said mammal an effective amount of the bacterium of    any one of embodiments 1 to 89, the composition of embodiment 90,    the pharmaceutical composition of embodiment 91, or the unit dosage    form of any one of embodiments 127 to 129.-   131. The method of embodiment 130, wherein said mammal is selected    from the group consisting of a human, a pig, a cow, and a sheep.-   132. The method of embodiment 130 or 131, wherein said bacterium,    said composition, said pharmaceutical composition, or said unit    dosage form is formulated for administration to said mammal.-   133. The method of any one of embodiments 130 to 132, wherein said    bacterium comprises an exogenous nucleic acid encoding a growth    factor.-   134. The method of embodiment 133, wherein said growth factor is    constitutively expressed in said bacterium.-   135. The method of embodiment 133 or 134, wherein said growth factor    is EGF.-   136. The method of embodiment 135, wherein said mammal is a pig and    said EGF is porcine EGF.-   137. A method of increasing binding of a bacterium to intestinal    mucosa comprising:    -   (a) contacting said microorganism with an exogenous nucleic acid        encoding a fusion protein, wherein said exogenous nucleic acid        encoding a fusion protein comprises a sequence encoding a CmbA        polypeptide; and    -   (b) expressing said exogenous nucleic acid encoding a fusion        protein in said bacterium.-   138. The method of embodiment 137, wherein said exogenous nucleic    acid encoding a fusion protein further comprises a sequence encoding    a TFF polypeptide.-   139. The method of embodiment 138, wherein expression of said    exogenous nucleic acid encoding a fusion protein produces a fusion    protein comprising said TFF and said CmbA.-   140. A kit comprising (1) a bacterium according to any one of    embodiments 1 to 89, a composition according to embodiment 90, a    pharmaceutical composition of embodiment 91, or a unit dosage form    of any one of embodiments 127 to 129, and (2) instructions for    administering said bacterium, said composition, said pharmaceutical    composition, or said unit dosage form to a mammal.-   141. The kit of embodiment 140, wherein said mammal is a human.-   142. A nucleic acid encoding a fusion protein, said nucleic acid    comprising:    -   (i) a sequence encoding a cell-adherence polypeptide selected        from the group consisting of cell and mucus-binding protein A        (CmbA), Mub, MapA, MbpL, and any combinations thereof; and    -   (ii) a sequence encoding a mucin-binding polypeptide selected        from a TFF polypeptide, MubBP, and a combination thereof.-   143. The nucleic acid of embodiment 142, wherein said mucin-binding    polypeptide is a TFF polypeptide.-   144. The nucleic acid of embodiment 143, wherein said TFF    polypeptide is TFF3.-   145. The nucleic acid of any one of embodiments 142 to 144, wherein    said cell-adherence polypeptide is CmbA.-   146. A plasmid comprising the nucleic acid of any one of embodiments    142 to 145.-   147. A bacterial host cell comprising the plasmid of embodiment 146.

What is claimed is:
 1. A recombinant lactic acid bacterium (LAB)comprising a first exogenous nucleic acid encoding a fusion protein,wherein the fusion protein comprises: (a) an N-terminal trefoil factor(TFF) polypeptide comprising an amino acid sequence at least 95%identical to SEQ ID NO: 3; and (b) a C-terminal cell adherencepolypeptide comprising a cell-wall anchoring domain, wherein the celladherence polypeptide is a cell and mucus-binding protein A (CmbA)comprising an amino acid sequence at least 95% identical to SEQ IDNO:
 1. 2. The recombinant LAB of claim 1, wherein said CmbA is aLactobacillus reuteri CmbA.
 3. The recombinant LAB of claim 1, whereinsaid TFF polypeptide is a human TFF3 polypeptide.
 4. The recombinant LABof claim 1, wherein said fusion protein further comprises a secretionsignal peptide.
 5. The recombinant LAB of claim 4, wherein saidsecretion signal peptide is a secretion leader of an unidentifiedsecreted 45-kDa protein (SSusp45).
 6. The recombinant LAB of claim 1,wherein said first exogenous nucleic acid is placed under the control ofa promoter selected from the group consisting of: a thyA promoter(PthyA), an hllA promoter (PhllA), and a gapB promoter (PgapB).
 7. Therecombinant LAB of claim 1, further comprising a second exogenousnucleic acid encoding at least one therapeutic polypeptide selected fromthe group consisting of: a) a cytokine; b) an interleukin (IL); c) anantibody or a functional fragment thereof; d) an antigen; e) a hormone;f) a TFF; and g) an enzyme or a functional fragment thereof.
 8. Therecombinant LAB of claim 7, wherein: a) said interleukin (IL) isselected from the group consisting of an IL-2, an IL-10, and an IL-18;b) said antibody is selected from the group consisting of an anti-TNFαantibody, an antibody to IL-4, an antibody to IL-5, an antibody toIL-13, an antibody to IL-15, and an antibody to immunoglobulin E (IgE);c) said antigen is selected from the group consisting of a proinsulin(PINS), a glutamic acid decarboxylase (GAD65), an insulinoma-associatedprotein 2 (IA-2), an islet-specific glucose-6-phosphatase catalyticsubunit-related protein (IGRP), a zinc transporter 8 (ZnT8), achromogranin A, a (prepro) islet amyloid polypeptide (ppIAPP), aperipherin, a citrullinated glucose-regulated protein (GRP), a treepollen allergen, a weed pollen allergen, a grass pollen allergen, a foodallergen, a dust-mite allergen, a mold allergen, an animal danderallergen; d) said hormone selected from the group consisting of aglucagon-like peptide 1 (GLP-1), a glucagon-like peptide 2 (GLP-2), aglucagon, an exendin-4, an epidermal growth factor, and a growthhormone; e) said TFF is selected from the group consisting of a TFF 1, aTFF2, and a TFF3; and f) said enzyme is selected from the groupconsisting of a phenylalanine ammonia lyase (PAL) and an amino aciddecarboxylase.
 9. The recombinant LAB of claim 8, wherein said at leastone therapeutic polypeptide is a PAL.
 10. The recombinant LAB of claim7, wherein said second exogenous nucleic acid is placed under thecontrol of a gapB promoter (PgapB).
 11. The recombinant LAB of claim 1,wherein said recombinant LAB is selected from the group consisting of aLactobacillus species (sp.) bacterium, a Bifidobacterium sp. bacterium,a Streptococcus sp. bacterium, an Enterococcus sp. bacterium, and aLactococcus sp. bacterium.
 12. The recombinant LAB of claim 11, whereinsaid LAB is a Lactococcus lactis.
 13. The recombinant LAB of claim 1,wherein said recombinant LAB has at least one feature that is differentfrom a corresponding LAB lacking said first exogenous nucleic acid,wherein said at least one feature is selected from the group of featuresconsisting of: a) an increased gastro-intestinal (GI) transit time of atleast 10%; b) an increased adherence to intestinal mucosa of at least10%; c) an increased in vitro mucin-binding capability of at least 20%;and d) an increased in vitro Caco-2 cell-binding capability of at least10%.
 14. The recombinant LAB of claim 13, wherein said recombinant LABhas an increased in vitro mucin-binding capability of at least 20% whencompared with said corresponding LAB lacking said first exogenousnucleic acid.
 15. The recombinant LAB of claim 13, wherein saidrecombinant LAB has an increased in vitro Caco-2 cell-binding capabilityof at least 10% when compared with said corresponding LAB lacking saidfirst exogenous nucleic acid.
 16. The recombinant LAB of claim 1,further comprising at least one mutation or insertion that increasestrehalose accumulation, wherein said at least one mutation or insertionis selected from the group consisting of: a) an insertion of a firstpolycistronic expression cassette comprising, in 5′ to 3′ order, a usp45promoter, a usp45 gene, an intergenic region that is immediately 5′ to arpmD gene, and a nucleic acid encoding an OtsB from Escherichia coli; b)an insertion of a second polycistronic expression cassette comprising,in 5′ to 3′ order, an hllA promoter (PhllA) and a nucleic acidcomprising trehalose transporter genes llmg_0453 and llmg_0454; c) aninactivated endogenous trehalose 6-phosphate phosphorylase gene (trePP),wherein said trePP gene is inactivated by gene deletion, so that saidrecombinant LAB lacks TrePP activity; and d) an inactivated endogenouscellobiose-specific PTS system IIC component (ptcC) gene, wherein saidptcC gene is inactivated by insertion of a premature stop codon, so thatsaid recombinant LAB lacks PtcC activity.
 17. A pharmaceuticalcomposition comprising said recombinant LAB of claim 1, and apharmaceutically acceptable carrier.
 18. The recombinant LAB of claim 1,wherein said LAB is a Lactococcus lactis.
 19. The recombinant LAB ofclaim 2, wherein said LAB is a Lactococcus lactis.
 20. The recombinantLAB of claim 3, wherein said LAB is a Lactococcus lactis.
 21. Therecombinant LAB of claim 4, wherein said LAB is a Lactococcus lactis.22. The recombinant LAB of claim 5, wherein said LAB is a Lactococcuslactis.
 23. The recombinant LAB of claim 6, wherein said LAB is aLactococcus lactis.
 24. The recombinant LAB of claim 7, wherein said LABis a Lactococcus lactis.
 25. The recombinant LAB of claim 8, whereinsaid LAB is a Lactococcus lactis.
 26. The recombinant LAB of claim 9,wherein said LAB is a Lactococcus lactis.
 27. The recombinant LAB ofclaim 10, wherein said LAB is a Lactococcus lactis.
 28. The recombinantLAB of claim 13, wherein said LAB is a Lactococcus lactis.
 29. Therecombinant LAB of claim 14, wherein said LAB is a Lactococcus lactis.30. The recombinant LAB of claim 15, wherein said LAB is a Lactococcuslactis.
 31. The recombinant LAB of claim 16, wherein said LAB is aLactococcus lactis.
 32. The pharmaceutical composition of claim 17,wherein said LAB is a Lactococcus lactis.